Engineered light-activated anion channel proteins and methods of use thereof

ABSTRACT

Aspects of the disclosure include compositions, devices, systems and methods for optogenetic modulation of action potentials in target cells. The subject devices include light-generating devices, control devices, and delivery devices for delivering light-responsive polypeptides, or nucleic acids encoding same, to target cells. The subject compositions and systems include light-activated polypeptides, nucleic acids comprising nucleotide sequences encoding these polypeptides, as well as expression systems that facilitate expression of these polypeptides in target cells. Also provided are methods of using the subject devices and systems to optogenetically manipulate action potentials in target cells, e.g., to treat a neurological or psychiatric condition in a human or animal subject.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 61/972,182, filed on Mar. 28,2014, the disclosure of which application is herein incorporated byreference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “STAN-1115WOSeqList_ST25.txt” created on Mar. 27, 2015 and having a size of 449 KB.The contents of the text file are incorporated by reference herein intheir entirety.

INTRODUCTION

Optogenetics refers to the combination of genetic and optical methodsused to control specific events in target cells with the temporalprecision (millisecond-timescale) needed to keep pace with functioningintact biological systems. Optogenetics involves the introduction offast light-responsive ion channel or pump proteins into the plasmamembranes of target cells to allow temporally precise manipulation ofmembrane potentials while maintaining cell-type resolution through theuse of specific targeting mechanisms, such as tissue-specific promoters.

Using light to silence electrical activity in target cells is a majorgoal of optogenetics. Available optogenetic proteins that directly moveions to achieve silencing are often inefficient, pumping only a singleion per photon across the cell membrane, rather than allowing many ionsper photon to flow through an ion channel pore.

There is a need in the art for improved light-responsive ion channels.

SUMMARY

Aspects of the disclosure include compositions, devices, systems andmethods for optogenetic modulation of action potentials in target cells.The subject devices include light-generating devices, control devices,and delivery devices for delivering light-responsive polypeptides, ornucleic acids encoding same, to target cells. The subject compositionsand systems include light-activated polypeptides, nucleic acidscomprising nucleotide sequences encoding these polypeptides, as well asexpression systems that facilitate expression of these polypeptides intarget cells. Also provided are methods of using the subject devices andsystems to optogenetically manipulate action potentials in target cells,e.g., to treat a neurological or psychiatric condition in a human oranimal subject.

The present disclosure provides a light-activated polypeptide comprisingan amino acid sequence that is at least 58% identical to SEQ ID NOS: 1,23, 34 or 56, wherein the polypeptide functions as a light-activatedanion channel. The present disclosure provides a nucleic acid comprisinga nucleotide sequence encoding a light-activated polypeptide comprisingan amino acid sequence that is at least 58% identical to SEQ ID NOS: 1,23, 34 or 56, wherein the polypeptide functions as a light-activatedanion channel. The present disclosure provides a recombinant expressionvector comprising a nucleic acid comprising a nucleotide sequenceencoding a light-activated polypeptide comprising an amino acid sequencethat is at least 58% identical to SEQ ID NOS: 1, 23, 34 or 56, whereinthe polypeptide functions as a light-activated anion channel. In somecases, the polypeptide functions as a light-activated chloride anionchannel. In some cases, the polypeptide comprises an amino acid sequencethat is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% identical, or is 100% identical, tothe amino acid sequence provided in SEQ ID NO:1 and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T98S, E129S,E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q, relative to theamino acid sequence of C1C2 (SEQ ID NO:78). In some cases, the first 50N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 167 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 195 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide comprises an amino acid sequence as provided in any oneof SEQ ID NOs: 1-22. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:34 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281K and/or N297Q,relative to the amino acid sequence of C1V1 (SEQ ID NO:80); in somecases, the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82); in somecases, the cysteine residue at position 167 is changed to a threonine,alanine or serine residue; in some cases, the aspartic acid residue atposition 195 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide comprises an amino acid sequence asprovided in any one of SEQ ID NOs: 34-55. In some cases, the polypeptidecomprises an amino acid sequence that is at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical, or is 100% identical, to the amino acid sequence provided inSEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T99S, E130S, E141S, E163S, V157K, H174R,A286N, P282K and/or N298Q, relative to the amino acid sequence of ReaChR(SEQ ID NO:81); in some cases, the first 51 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82); in some cases, the cysteine residue at position 168 ischanged to a threonine, alanine or serine residue; in some cases, theaspartic acid residue at position 196 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide comprises anamino acid sequence as provided in any one of SEQ ID NOs: 56-77. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from A59S, E90S, E101S, E123S,Q117K, H134R, V242K, T246N and/or N258Q, relative to the amino acidsequence of ChR2 (SEQ ID NO:79); in some cases, the cysteine residue atposition 128 is changed to a threonine, alanine or serine residue; insome cases, the aspartic acid residue at position 156 is changed to analanine residue or an asparagine residue. In some cases, the polypeptidecomprises an amino acid sequence as provided in any one of SEQ ID NOs:23-33.

The present disclosure provides a pharmaceutical composition comprising;a) a nucleic acid comprising a nucleotide sequence encoding alight-activated polypeptide as described above, and elsewhere herein;and b) a pharmaceutically acceptable carrier. The present disclosureprovides a pharmaceutical composition comprising; a) a light-activatedpolypeptide as described above, and elsewhere herein; and b) apharmaceutically acceptable carrier.

The present disclosure provides a cell comprising a nucleic acidcomprising a nucleotide sequence encoding a light-activated polypeptideas described above, and elsewhere herein. The present disclosureprovides a cell comprising a recombinant expression vector comprising anucleic acid comprising a nucleotide sequence encoding a light-activatedpolypeptide as described above, and elsewhere herein.

The present disclosure provides a system for modulating the membranepotential of a cell, the system comprising: a nucleic acid encoding apolypeptide that comprises an amino acid sequence that is at least 58%identical to SEQ ID NOs: 1, 23, 34 or 56, wherein the polypeptidefunctions as a light-activated anion channel; and a device configured toilluminate a target location with light. In some cases, the polypeptidefunctions as a light-activated chloride ion channel. In some cases, thepolypeptide comprises an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical, or is 100% identical, to the amino acid sequenceprovided in SEQ ID NO:1 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 aminoacid substitutions selected from T98S, E129S, E140S, E162S, V156K,H173R, T285N, V281K and/or N297Q, relative to the amino acid sequence ofC1C2 (SEQ ID NO:78). In some cases, the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82). In some cases, the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue. In some cases, theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide comprises anamino acid sequence as provided in any one of SEQ ID NOs: 1-22. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from T98S, E129S, E140S, E162S,V156K, H173R, A285N, P281K and/or N297Q, relative to the amino acidsequence of C1V1 (SEQ ID NO:80). In some cases, the first 50 N-terminalamino acid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. Insome cases, the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue. In some cases, the polypeptidehas an amino acid sequence as provided in any one of SEQ ID NOs: 34-55.In some cases, the polypeptide comprises an amino acid sequence that isat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identical, or is 100% identical, identical tothe amino acid sequence provided in SEQ ID NO:56 and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T99S, E130S,E141S, E163S, V157K, H174R, A286N, P282K and/or N298Q, relative to theamino acid sequence of ReaChR (SEQ ID NO:81). In some cases, the first51 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 196 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 56-77. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, identical to the amino acid sequence provided in SEQ ID NO:23and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246Nand/or N258Q, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue or anasparagine residue. In some cases, the polypeptide comprises an aminoacid sequence as provided in any one of SEQ ID NOs: 23-33. In somecases, the device is configured to illuminate the target location withlight having a wavelength ranging from about 350 to about 750 nm. Insome cases, the device is configured to illuminate the target locationwith light having a wavelength ranging from about 450 up to about 500nm. In some cases, the device is configured to constantly illuminate thetarget location with light. In some cases, the device is configured toilluminate the target location with pulses of light. In some cases, thedevice is configured to modulate the wavelength and/or the intensity ofthe light. In some cases, the device is configured to modulate thefrequency and/or the duration of the pulses of light. In some cases, thedevice is configured to illuminate the target location in response to auser input. In some cases, the user input comprises: the wavelength oflight, the intensity of light, the duration of a light pulse, thefrequency of a light pulse, and/or the target location. In some cases,the device is adapted to be implanted in a subject. In some cases, thetarget location is: a cell, a portion of a cell, a plurality of cells, abundle of nerve fibers, a neuromuscular junction, a central nervoussystem (CNS) tissue, a peripheral nervous system (PNS) tissue, or ananatomical region.

The present disclosure provides a method for modulating the membranepotential of a cell in response to light, the method comprising:exposing a cell to light of an activating wavelength, wherein the cellis genetically modified with a nucleic acid encoding a polypeptide thatcomprises an amino acid sequence that is at least 58% identical to SEQID NOs: 1, 23, 34 or 56, wherein the polypeptide functions as alight-activated anion channel. In some cases, the polypeptide comprisesan amino acid sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identical, oris 100% identical, to the amino acid sequence provided in SEQ ID NO: 1and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281Kand/or N297Q, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue or anasparagine residue. In some cases, the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 1-22. In some cases, thepolypeptide comprises an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical, or is 100% identical, to the amino acid sequenceprovided in SEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acid substitutions selected from T98S, E129S, E140S, E162S, V156K,H173R, A285N, P281K and/or N297Q, relative to the amino acid sequence ofC1V1 (SEQ ID NO:80). In some cases, the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82). In some cases, the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue. In some cases, theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 34-55. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from T99S, E130S, E141S, E163S,V157K, H174R, A286N, P282K and/or N298Q, relative to the amino acidsequence of ReaChR (SEQ ID NO:81). In some cases, the first 51N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 196 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide comprises an amino acid sequence as provided in any oneof SEQ ID NOs: 56-77. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:23 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q,relative to the amino acid sequence of ChR2 (SEQ ID NO:79). In somecases, the cysteine residue at position 128 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 156 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide comprises an amino acid sequence asprovided in any one of SEQ ID NOs: 23-33.

The present disclosure provides a method of treating a condition in asubject, the method comprising genetically modifying a target cell ofthe subject with a nucleic acid comprising a nucleotide sequenceencoding a polypeptide that comprises an amino acid sequence that is atleast 58% identical to SEQ ID NOs: 1, 23, 34 or 56, wherein thepolypeptide functions as a light-activated anion channel; and exposingthe target cell to light of an activating wavelength to treat thesubject for the condition. In some cases, the polypeptide comprises anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identical, or is100% identical, to the amino acid sequence provided in SEQ ID NO:1 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q,relative to the amino acid sequence of C1C2 (SEQ ID NO:78). In somecases, the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82). In somecases, the cysteine residue at position 167 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 195 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide comprises an amino acid sequence asprovided in any one of SEQ ID NOs: 1-22. In some cases, the polypeptidecomprises an amino acid sequence that is at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical, or is 100% identical, to the amino acid sequence provided inSEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,A285N, P281K and/or N297Q, relative to the amino acid sequence of C1V1(SEQ ID NO:80). In some cases, the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82). In some cases, the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue. In some cases, theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide comprises anamino acid sequence as provided in any one of SEQ ID NOs: 34-55. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from T99S, E130S, E141S, E163S,V157K, H174R, A286N, P282K and/or N298Q, relative to the amino acidsequence of ReaChR (SEQ ID NO:81). In some cases, the first 51N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 196 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide comprises an amino acid sequence as provided in any oneof SEQ ID NOs: 56-77. In some cases, the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:23 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q,relative to the amino acid sequence of ChR2 (SEQ ID NO:79). In somecases, the cysteine residue at position 128 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 156 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide has an amino acid sequence as provided inany one of SEQ ID NOs: 23-33. In some cases, the target cell is a nervecell.

The present disclosure provides a method of inhibiting the formation ofan action potential in a nerve cell or a portion thereof, the methodcomprising: genetically modifying the nerve cell with a nucleic acidcomprising a nucleotide sequence encoding a polypeptide that comprisesan amino acid sequence that is at least 58% identical to SEQ ID NOs: 1,23, 34 or 56, wherein the polypeptide functions as a light-activatedanion channel; and exposing at least a portion of the nerve cell tolight of an activating wavelength to inhibit the formation of an actionpotential in the nerve cell or in a portion thereof. In some cases, thepolypeptide comprises an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical, or is 100% identical, to the amino acid sequenceprovided in SEQ ID NO:1 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 aminoacid substitutions selected from T98S, E129S, E140S, E162S, V156K,H173R, T285N, V281K and/or N297Q, relative to the amino acid sequence ofC1C2 (SEQ ID NO:78). In some cases, the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82). In some cases, the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue. In some cases, theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide comprises anamino acid sequence as provided in any one of SEQ ID NOs: 1-22. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from T98S, E129S, E140S, E162S,V156K, H173R, A285N, P281K and/or N297Q, relative to the amino acidsequence of C1V1 (SEQ ID NO:80). In some cases, the first 50 N-terminalamino acid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. Insome cases, the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue. In some cases, the polypeptidecomprises an amino acid sequence as provided in any one of SEQ ID NOs:34-55. In some cases, the polypeptide comprises an amino acid sequencethat is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% identical, or is 100% identical, tothe amino acid sequence provided in SEQ ID NO:56 and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T99S, E130S,E141S, E163S, V157K, H174R, A286N, P282K and/or N298Q, relative to theamino acid sequence of ReaChR (SEQ ID NO:81). In some cases, the first51 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 196 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 56-77. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:23 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q,relative to the amino acid sequence of ChR2 (SEQ ID NO:79). In somecases, the cysteine residue at position 128 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 156 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide comprises an amino acid sequence asprovided in any of SEQ ID NOs: 23-33.

The present disclosure provides a kit comprising: a nucleic acidencoding a polypeptide that is at least 58% identical to SEQ ID NOs: 1,23, 34 or 56, wherein the polypeptide functions as a light-activatedanion channel. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:1 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q,relative to the amino acid sequence of C1C2 (SEQ ID NO:78). In somecases, the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82). In somecases, the cysteine residue at position 167 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 195 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide comprises an amino acid sequence asprovided in any one of SEQ ID NOs: 1-22. In some cases, the polypeptidecomprises an amino acid sequence that is at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical, or is 100% identical, to the amino acid sequence provided inSEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,A285N, P281K and/or N297Q, relative to the amino acid sequence of C1V1(SEQ ID NO:80). In some cases, the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82). In some cases, the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue. In some cases, theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue. In some cases, the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 34-55. In somecases, the polypeptide comprises an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99% identical, or is 100% identical, to the amino acidsequence provided in SEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid substitutions selected from T99S, E130S, E141S, E163S,V157K, H174R, A286N, P282K and/or N298Q, relative to the amino acidsequence of ReaChR (SEQ ID NO:81). In some cases, the first 51N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 196 ischanged to an alanine residue or an asparagine residue. In some cases,the polypeptide comprises an amino acid sequence as provided in any oneof SEQ ID NOs: 56-77. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:23 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q,relative to the amino acid sequence of ChR2 (SEQ ID NO:79). In somecases, the cysteine residue at position 128 is changed to a threonine,alanine or serine residue. In some cases, the aspartic acid residue atposition 156 is changed to an alanine residue or an asparagine residue.In some cases, the polypeptide has an amino acid sequence as provided inany one of SEQ ID NOs: 23-33. In some cases, kit further comprises adevice configured to illuminate a target location with a light. In somecases, the device is configured to illuminate the target location withlight having a wavelength ranging from about 350 to about 750 nm. Insome cases, the device is configured to illuminate the target locationwith light having a wavelength ranging from about 450 up to about 500nm. In some cases, the device is configured to constantly illuminate thetarget location with a light. In some cases, the device is configured toilluminate the target location with pulses of light. In some cases, thedevice is configured to modulate the wavelength and/or the intensity ofthe light. In some cases, the device is configured to modulate thefrequency and/or duration of the pulses of light. In some cases, thedevice is configured to illuminate the target location in response to auser input. In some cases, the user input comprises: the wavelength oflight, the intensity of light, the duration of a light pulse, thefrequency of a light pulse, and/or the target location to be illuminatedby the light. In some cases, the device is adapted to be implanted in asubject. In some cases, the target location is: a cell, a portion of acell, a plurality of cells, a bundle of nerve fibers, a neuromuscularjunction, a CNS tissue, a PNS tissue, or an anatomical region.

The present disclosure provides a light-activated polypeptide comprisingan amino acid sequence that is at least 60% identical to SEQ ID NOS: 94,116, 127 or 149, wherein the polypeptide functions as a light-activatedanion channel. The present disclosure provides a nucleic acid comprisinga nucleotide sequence encoding a light-activated polypeptide comprisingan amino acid sequence that is at least 60% identical to SEQ ID NOS: 94,116, 127 or 149, wherein the polypeptide functions as a light-activatedanion channel. The present disclosure provides a recombinant expressionvector comprising a nucleic acid comprising a nucleotide sequenceencoding a light-activated polypeptide comprising an amino acid sequencethat is at least 60% identical to SEQ ID NOS: 94, 116, 127 or 149,wherein the polypeptide functions as a light-activated anion channel. Insome cases, the polypeptide functions as a light-activated chlorideanion channel. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:94 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, V281R, T285N,N297Q and/or E312S, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 94-115. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163 S, P282R, A286N,N298Q and/or E313 S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 149-170. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 116-126.

The present disclosure provides a pharmaceutical composition comprising:a) a nucleic acid comprising a nucleotide sequence encoding alight-activated polypeptide comprising an amino acid sequence that is atleast 60% identical to SEQ ID NOS: 94, 116, 127 or 149, wherein thepolypeptide functions as a light-activated anion channel; and b) apharmaceutically acceptable carrier.

The present disclosure provides a cell comprising a nucleic acidcomprising a nucleotide sequence encoding a light-activated polypeptidecomprising an amino acid sequence that is at least 60% identical to SEQID NOS: 94, 116, 127 or 149, wherein the polypeptide functions as alight-activated anion channel. The present disclosure provides a cellcomprising a recombinant expression vector comprising a nucleotidesequence encoding a light-activated polypeptide comprising an amino acidsequence that is at least 60% identical to SEQ ID NOS: 94, 116, 127 or149, wherein the polypeptide functions as a light-activated anionchannel.

The present disclosure provides a system for modulating the membranepotential of a cell, the system comprising: a) a nucleic acid comprisinga nucleotide sequence encoding a polypeptide that comprises an aminoacid sequence that is at least 60% identical to SEQ ID NOs: 94, 116, 127or 149, wherein the polypeptide functions as a light-activated anionchannel; and b) a device configured to illuminate a target location withlight. In some cases, the polypeptide functions as a light-activatedchloride ion channel. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:94 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, V281R, T285N,N297Q and/or E312S, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 94-115. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163S, P282R, A286N,N298Q and/or E313S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 149-170. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 116-126. In some cases, the device is configured toilluminate the target location with light having a wavelength rangingfrom about 350 to about 750 nm. In some cases, the device is configuredto illuminate the target location with light having a wavelength rangingfrom about 450 up to about 500 nm. In some cases, the device isconfigured to constantly illuminate the target location with light. Insome cases, the device is configured to illuminate the target locationwith pulses of light. In some cases, the device is configured tomodulate the wavelength and/or the intensity of the light. In somecases, the device is configured to modulate the frequency and/or theduration of the pulses of light. In some cases, the device is configuredto illuminate the target location in response to a user input. In somecases, the user input comprises: the wavelength of light, the intensityof light, the duration of a light pulse, the frequency of a light pulse,and/or the target location. In some cases, the device is adapted to beimplanted in a subject. In some cases, the target location is: a cell, aportion of a cell, a plurality of cells, a bundle of nerve fibers, aneuromuscular junction, a CNS tissue, a PNS tissue, or an anatomicalregion.

The present disclosure provides a method for modulating the membranepotential of a cell in response to light, the method comprising:exposing a cell to light of an activating wavelength, wherein the cellis genetically modified with a nucleic acid comprising a nucleotidesequence encoding a polypeptide that comprises an amino acid sequencethat is at least 58% identical to SEQ ID NOs: 94, 116, 127 or 149,wherein the polypeptide functions as a light-activated anion channel. Insome cases, the polypeptide comprises an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identical, or is 100% identical, to the aminoacid sequence provided in SEQ ID NO:94 and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 amino acid substitutions selected from T98S, E122N, E129Q,E140S, V156R, E162S, V281R, T285N, N297Q and/or E312S, relative to theamino acid sequence of C1C2 (SEQ ID NO:78). In some cases, the first 50N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteineresidue at position 167 is changed to a threonine, alanine or serineresidue. In some cases, the aspartic acid residue at position 195 ischanged to an alanine residue, an asparagine residue, or a cysteineresidue. In some cases, the aspartic acid residue at position 156 ischanged to an alanine residue, an asparagine residue, or a cysteineresidue. In some cases, the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 94-115. In some cases, thepolypeptide comprises an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% identical, or is 100% identical, to the amino acid sequenceprovided in SEQ ID NO: 127 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T98S, E122N, E129Q, E140S, V156R,E162S, P281R, A285N, N297Q and/or E312S, relative to the amino acidsequence of C1V1 (SEQ ID NO:80). In some cases, the first 50 N-terminalamino acid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. Insome cases, the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue. In somecases, the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue. In somecases, the polypeptide has an amino acid sequence as provided in any oneof SEQ ID NOs: 127-148. In some cases, the polypeptide comprises anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identical, or is100% identical, to the amino acid sequence provided in SEQ ID NO:149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163S, P282R, A286N,N298Q and/or E313S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 149-170. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273 S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 116-126.

The present disclosure provides a method of treating a condition in asubject, the method comprising: a) genetically modifying a target cellof the subject with a nucleic acid comprising a nucleotide sequenceencoding a polypeptide that comprises an amino acid sequence that is atleast 60% identical to SEQ ID NOs: 94, 116, 127 or 149, wherein thepolypeptide functions as a light-activated anion channel; and b)exposing the target cell to light of an activating wavelength to treatthe subject for the condition. In some cases, the polypeptide comprisesan amino acid sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identical, oris 100% identical to the amino acid sequence provided in SEQ ID NO:94and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, V281R, T285N,N297Q and/or E312S, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 94-115. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163S, P282R, A286N,N298Q and/or E313S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 149-170. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 116-126. In some cases, the target cell is a nerve cell.

The present disclosure provides a method of inhibiting the formation ofan action potential in a nerve cell or a portion thereof, the methodcomprising: a) genetically modifying the nerve cell with a nucleic acidcomprising a nucleotide sequence encoding a polypeptide that comprisesan amino acid sequence that is at least 60% identical to SEQ ID NOs: 94,116, 127 or 149, wherein the polypeptide functions as a light-activatedanion channel; and b) exposing at least a portion of the nerve cell tolight of an activating wavelength to inhibit the formation of an actionpotential in the nerve cell or in a portion thereof. In some cases, thepolypeptide comprises an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO:94 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T98S, E122N, E129Q, E140S, V156R,E162S, V281R, T285N, N297Q and/or E312S, relative to the amino acidsequence of C1C2 (SEQ ID NO:78). In some cases, the first 50 N-terminalamino acid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82). In some cases, the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. Insome cases, the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue. In somecases, the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue. In somecases, the polypeptide comprises an amino acid sequence as provided inany one of SEQ ID NOs: 94-115. In some cases, the polypeptide comprisesan amino acid sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identical, oris 100% identical, to the amino acid sequence provided in SEQ ID NO:127and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 127-148. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163 S, P282R, A286N,N298Q and/or E313 S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 149-170. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any of SEQ ID NOs:116-126.

The present disclosure provides a kit comprising: a nucleic acidencoding a polypeptide that is at least 60% identical to SEQ ID NOs: 94,116, 127 or 149, wherein the polypeptide functions as a light-activatedanion channel. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:94 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, V281R, T285N,N297Q and/or E312S, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 94-115. In some cases, the polypeptide comprises an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical, or is 100%identical to the amino acid sequence provided in SEQ ID NO:127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80). In some cases, the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 167 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 195 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 127-148. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO:149 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163 S, P282R, A286N,N298Q and/or E313 S, relative to the amino acid sequence of ReaChR (SEQID NO:81). In some cases, the first 51 N-terminal amino acid residuesare replaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82). In some cases, the cysteine residue at position 168 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 196 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide comprises an amino acid sequence as provided in any one ofSEQ ID NOs: 149-170. In some cases, the polypeptide comprises an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identical, or is 100%identical, to the amino acid sequence provided in SEQ ID NO: 116 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Qand/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79). In some cases, the cysteine residue at position 128 is changedto a threonine, alanine or serine residue. In some cases, the asparticacid residue at position 156 is changed to an alanine residue, anasparagine residue, or a cysteine residue. In some cases, thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 116-126. In some cases, the kit further comprises a deviceconfigured to illuminate a target location with a light. In some cases,the device is configured to illuminate the target location with lighthaving a wavelength ranging from about 350 to about 750 nm. In somecases, the device is configured to illuminate the target location withlight having a wavelength ranging from about 450 up to about 500 nm. Insome cases, the device is configured to constantly illuminate the targetlocation with a light. In some cases, the device is configured toilluminate the target location with pulses of light. In some cases, thedevice is configured to modulate the wavelength and/or the intensity ofthe light. In some cases, the device is configured to modulate thefrequency and/or duration of the pulses of light. In some cases, thedevice is configured to illuminate the target location in response to auser input. In some cases, the user input comprises: the wavelength oflight, the intensity of light, the duration of a light pulse, thefrequency of a light pulse, and/or the target location to be illuminatedby the light. In some cases, the device is adapted to be implanted in asubject. In some cases, the target location is: a cell, a portion of acell, a plurality of cells, a bundle of nerve fibers, a neuromuscularjunction, a CNS tissue, a PNS tissue, or an anatomical region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panels A-E show details of the rational design and screen:V_(rev)-shifted ChRs. (Panel A) C1C2 crystal structure with residuesused for screening. (Panel B) C1C2 mutations screened in neurons forphotocurrent size at −80 mV (n=6-8). Arrows: nine mutations selected forC1C2_5x (T98S/E129S/E140S/E162S/T285N) and C1C2_4x(V156K/H173R/V281K/N297Q) constructs. (Panel C) V_(rev) of C1C2single-mutation constructs (n=6-8). (Panel D) Comparison of photocurrentsizes for C1C2, C1C2_4x and C1C2_5x. (Panel E) Comparison of V_(rev) forC1C2, C1C2_4x and C1C2_5x (n=8-10). Error bars: s.e.m.

FIG. 2, Panels A-J show biophysical properties of iC1C2. (Panel A) C1C2structure with the nine residues mutated in C1C2_4x and C1C2_5x. (PanelB) Representative photocurrents and (Panel C) correspondingcurrent-voltage relationships recorded at membrane potentials from −75mV to +55 mV upon 475 nm light activation (power density: 5 mW/mm²).(Panel D) V_(rev) of C1C2, iC1C2, C1C2_4x, and C1C2_5x (neuronalrecording solutions in Methods). (Panel E) Activation spectra of NpHR,C1C2, and iC1C2 measured at power density 0.65 mW/mm² for eachwavelength and normalized to the maximum amplitude (n=6). (Panel F)V_(rev) of C1C2, iC1C2, C1C2_4x, and C1C2_5x with internal (int) 120 mMKCl and external (ext) 120 mM NaCl, CsCl, or Na-Gluconate, pH 7.3,characterized in HEK cells. (Panel G) As in (Panel A), with ext 120 mMNaCl and int 120 mM KCl, CsCl, or K-Gluconate, pH 7.3 (n=6-17). (PanelH) V_(rev) of iC1C2 under equal (eq) external and internal pH,generating a Nernst potential for protons of 0 mV (upper dotted line) atpH 6 and 7.3. [Cl⁻]_(i) concentration was 8 mM and [Cl⁻]₀ was 128 mM,generating a Nernst potential for Cl⁻ of −71 mV (lower dotted line)(n=6-9). (Panel I) Current-voltage relationship recorded with equalexternal and internal pH values at pH 6 and 7.3; internal and external[Cl] of 8 mM and 128 mM, respectively (n=3-8). (Panel J) Photocurrent ofiC1C2 at 0 mV from the current-voltage relationship in (Panel I). Errorbars: s.e.m.

FIG. 3, Panels A-F show data relating to characterization of iC1C2 inmammalian neurons. (Panel A) Representative photocurrents of C1C2 (left)and iC1C2 (right) recorded at membrane potentials ranging from −80 to 0mV 475 nm light (“475 nm” bar) was applied at 5 mW/mm². (Panel B)Corresponding current-voltage relationship for photocurrents. (Panel C)V_(rev) of C1C2 and iC1C2 relative to threshold for spike generation(V_(AP)) and to neuron resting potential (V_(rest)) (n=8-22). (Panel D)Photocurrent amplitudes (left) and membrane polarization at V_(AP)(right) (n=9-14). (Panel E) Mean changes in input resistances werenormalized to pre-light value (n=9-20). (Panel F) Sample voltage tracesof iC1C2-expressing neuron stimulated with varying current injectionsfor 1.5 s, and additional 0.5 s, 475 nm, 5 mW/mm² pulses showingeffective clamping toward V_(rev): note hyperpolarizing responses belowV_(A). Error bars: s.e.m.

FIG. 4, Panels A-H show data relating to fast and bistable inhibition ofneuronal spiking with iC1C2 and SwiChR. Representative voltage traces ofiC1C2-expressing neurons stimulated with either (Panel A) a continuouselectrical pulse (3 s) or (Panel B) pulsed current injections (10 Hz/3s). Electrically evoked spikes were inhibited by 475 nm light (“475 nm”bar) at 5 mW/mm². (Panel C) Distribution of spike-inhibition probabilityfor iC1C2-expressing cells (n=18 neurons; fraction of spikes blockedshown). (Panel D) Inward and outward photocurrents of SwiChR_(CT) in HEKcell upon 475 nm light (“475 nm” bar). (Panel E) Channel off-kinetics(τ) for iC1C2, SwiChR_(CT), and SwiChR_(CT) exposed to red light duringchannel closure. (Panel F) Light sensitivity of SwiChR_(CT) compared toiC1C2 and NpHR. iC1C2 and SwiChR_(CT) were activated with 470 nm andNpHR with 560 nm. Photocurrents were measured at light intensitiesbetween 0.0036 and 5 mW/mm², holding potential was −80 mV. Amplitudeswere normalized to the maximum value for each construct (n=6-8). (PanelG) Reversal potential of iC1C2 and iC1C2-C167T relative to VAP andV_(rest) (n=10-22) (left). Photocurrent amplitudes at VAP shown at right(n=9-15). (Panel H) Bistable spiking modulation by SwiChR_(CT). Spikingwas induced by a continuous electrical pulse (3 s) and stably inhibitedby 475 nm light (“475 nm” bar). Spiking resumed after 632 nm lightapplication (“632 nm” bar). Light power density was 5 mW/mm². Errorbars: s.e.m.

FIG. 5 shows a protein sequence alignment of the proteins C1C2 (SEQ IDNO:78) and iC1C2 (SEQ ID NO: 1) to ChR2 (SEQ ID NO:79). The mutatedresidues are shown in boxes, and the transmembrane helices are indicatedwith the bars labeled TM 1-7. Positioning of the SwiChR mutations isindicated by dashed ellipse.

FIG. 6 shows confocal microscope images of cultured neurons expressingC1C2-eYFP (left panel) and iC1C2-eYFP (right panel). Scale bar: 20 μm.

FIG. 7, Panels A-D show bistable inhibition with SwiChRs. (Panel A)Bistable spiking modulation with SwiChR_(CT). Spiking was induced by acontinuous electrical pulse (3 s), stably inhibited by 475 nm light(“475 nm” bar) delivered at 5 mW/mm², and resumed after 632 nm lightapplication (“632 nm” bar) (left). Prolonged spiking modulation in thesame cell after only 475 nm light delivery (“475 nm” bar) at 5 mW/mm²(right) with the same current injection as in left panel. (Panel B)Current-voltage relationship of SwiChR_(CA) recorded at membranepotentials from −80 mV to −30 mV upon 470 nm activation pulses (“475 nm”bar) followed by 632 nm (“632 nm” bar) light pulses. (Panel C)Representative photocurrent of SwiChR_(CA) upon 470 nm activation (“475nm” bar) followed by a second light pulse at 632 nm (“632 nm” bar).Light power density was 5 mW/mm². (Panel D) (left) Bistable spikingmodulation by SwiChR_(CA). Spiking was induced by a continuouselectrical pulse (3 s) and stably inhibited by 475 nm light (“475 nm”bar) delivered at 5 mW/mm². Spiking resumed after 632 nm lightapplication (“632 nm” bar). (Panel E) Prolonged spiking modulation ofSwiChR_(CA) of the same cell as in (Panel D) with 475 nm light (“475 nm”bar) only, delivered at 5 mW/mm²; the same current injection was used toinduce spiking as in (Panel D).

FIG. 8 shows a first example of an optical stimulation system inaccordance with embodiments of the disclosure.

FIG. 9 shows a second example of an optical stimulation system inaccordance with embodiments of the disclosure.

FIG. 10 shows a third example of an optical stimulation system inaccordance with embodiments of the disclosure.

FIG. 11 shows a flow diagram that illustrates the steps of an examplemethod in accordance with embodiments of the disclosure.

FIGS. 12-16 provide amino acid sequences of examples of polypeptidesthat relate to the compositions of the disclosure.

FIG. 17, Panels A-B show four graphs that demonstrate the reversalpotential and activity of improved inhibitory channelrhodopsins measuredin cultured neurons. Panel A, left: The reversal potential of ibC1C2_3.0(SEQ ID NO: 96) is −79 mV at 4 mM internal chloride concentration whichis lower than the threshold for action potentials (VAP) and the restingpotential of neurons (VRest). Panel A, Right: Outward currents at VAPcompared to regular ibC1C2 (SEQ ID NO: 3). Panel B, Left: At 12 mMinternal chloride, the reversal potentials ofibC1C2_3.0 (SEQ ID NO: 96)and SwiChR_3.0 (SEQ ID NO: 108) overlap with the equilibrium potentialfor chloride (VEq). Panel B, Right: Photocurrents of ibC1C2_3.0 (SEQ IDNO: 96) are 7 times larger compared to ibC1C2 (SEQ ID NO: 3). (Errorbars=standard error of the mean, number of recordings ≧6).

FIG. 18, Panels A-C show three graphs that demonstrate inhibition ofcultured neurons. Panel A: A 4 s light pulse (475 nm, 5 mW/mm²) inhibitsaction potentials in an ibC1C2_3.0 (SEQ ID NO: 96)-expressing neuronduring a 6 second period of electrical stimulations at 10 Hz. Panel B:Spike inhibition probability at 4 mM and (Panel C) 12 mM internalchloride. (Error bars=standard error of the mean, number of recordings≧6).

FIG. 19, Panels A-C show graphs that demonstrate inhibition of strong,extended stimulations in cultured neurons at 12 mM internal chloride.Panel A: Inhibition of an ibC1C2_3.0 (SEQ ID NO: 96)-expressing neuronwith a 10 s blue light pulse (475 nm, 5 mW/mm²) during a 12 secondperiod of electrical stimulation at 20 Hz. Panel B: Bimodal inhibitionof a SwiChR_3.0 (SEQ ID NO: 108)-expressing neuron: Electricalstimulations were applied for 16 seconds at 20 Hz. After 1 s, a briefblue light pulse (1 s, 475 nm, 5 mW/mm²) inhibits action potentials for13 seconds. Subsequently, a red light pulse (2 s, 632 nm, 10 mW/mm²)recovers action potential generation. Panel C: Spike inhibitionprobability for both constructs under these conditions. (Errorbars=standard error of the mean, number of recordings ibC1C2_3.0 (SEQ IDNO: 96): n=15, SwiChR_3.0 (SEQ ID NO: 108): n=5).

FIG. 20 shows a collection of graphs that demonstrate inhibition of aSwiChR_3.0 (SEQ ID NO: 108)-expressing neuron for 1 min at 12 mMinternal chloride concentration.

FIGS. 21-24 provide amino acid sequences of examples of polypeptidesthat relate to the compositions of the disclosure.

DEFINITIONS

As used herein, an “individual,” “subject,” or “patient” can be amammal, including a human. Mammals include, but are not limited to,ungulates, canines, felines, bovines, ovines, non-human primates,lagomorphs, and rodents (e.g., mice and rats). In one aspect, anindividual is a human. In another aspect, an individual is a non-humanmammal.

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxyl grouppresent at the carboxyl terminus of a polypeptide. In keeping withstandard polypeptide nomenclature, J. Biol. Chem., 243 (1969), 3552-59is used.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxynucleotides. Thus, this term includes, but isnot limited to, single-, double-, or multi-stranded DNA or RNA, genomicDNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”). Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid by endonucleases, in a form not normally found in nature. Thus anisolated nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

Nucleic acid sequence identity (as well as amino acid sequence identity)is calculated based on a reference sequence, which may be a subset of alarger sequence, such as a conserved motif, coding region, flankingregion, etc. A reference sequence will usually be at least about 18residues long, more usually at least about 30 residues long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990), J. Mol. Biol. 215:403-10 (using defaultsettings, i.e. parameters w=4 and T=17). As used herein, an amino acidsequence that is “at least x % identical” shares at least x % amino acidsequence identity with a reference sequence. For example, as usedherein, an amino acid sequence that is “at least 99% identical” sharesat least 99% amino acid sequence identity with a reference sequence.

The term “genetic modification” and refers to a permanent or transientgenetic change induced in a cell following introduction into the cell ofnew nucleic acid (i.e., nucleic acid exogenous to the cell). Geneticchange (“modification”) can be accomplished by incorporation of the newnucleic acid into the genome of the host cell, or by transient or stablemaintenance of the new nucleic acid as an extrachromosomal element.Where the cell is a eukaryotic cell, a permanent genetic change can beachieved by introduction of the nucleic acid into the genome of thecell. Suitable methods of genetic modification include viral infection,transfection, conjugation, protoplast fusion, electroporation, particlegun technology, calcium phosphate precipitation, direct microinjection,and the like.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, or a cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, or thecell naturally occurs. An isolated genetically modified host cell may bepresent in a mixed population of genetically modified host cells. Anisolated polypeptide will in some embodiments be synthetic. “Syntheticpolypeptides” are assembled from amino acids, and are chemicallysynthesized in vitro, e.g., cell-free chemical synthesis, usingprocedures known to those skilled in the art.

Amino acid substitutions in a native protein sequence may be“conservative” or “non-conservative” and such substituted amino acidresidues may or may not be one encoded by the genetic code. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a chemicallysimilar side chain (i.e., replacing an amino acid possessing a basicside chain with another amino acid with a basic side chain). A“non-conservative amino acid substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a chemicallydifferent side chain (i.e., replacing an amino acid having a basic sidechain with an amino acid having an aromatic side chain). The standardtwenty amino acid “alphabet” is divided into chemical families based onchemical properties of their side chains. These families include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) and sidechains having aromatic groups (e.g., tyrosine, phenylalanine,tryptophan, histidine).

As used herein, the term “light-activated protein” means a protein thatundergoes a conformational change when exposed to light of an activatingwavelength.

As used herein, the term “anion channel protein” means a protein thatincludes an anion pore that can be opened to allow a stream of anions topass from one side of a cell membrane to the other.

As used herein, an “effective dosage” or “effective amount” of a drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. An effectivedosage can be administered in one or more administrations. For purposesof this disclosure, an effective dosage of a drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this disclosure, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, delaying the progression of the disease,and/or prolonging survival of individuals.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing various aspects of embodiments of the invention ingreater detail, aspects of the systems and devices of variousembodiments are reviewed first in greater detail, followed by adiscussion of methods and kits according to certain embodiments of theinvention.

DETAILED DESCRIPTION

Aspects of the disclosure include compositions, devices, systems andmethods for optogenetic modulation of action potentials in target cells.The subject devices include light-generating devices, control devices,and delivery devices for delivering light-responsive polypeptides, ornucleic acids encoding same, to target cells. The subject compositionsand systems include light-activated polypeptides, nucleic acidscomprising nucleotide sequences encoding these polypeptides, as well asexpression systems that facilitate expression of these polypeptides intarget cells. Also provided are methods of using the subject devices andsystems to optogenetically manipulate action potentials in target cells,e.g., to treat a neurological or psychiatric condition in a human oranimal subject.

Compositions

Aspects of the disclosure include compositions for optogeneticallymodulating action potentials in target cells. The subject compositionsgenerally include an engineered light-activated anion channel proteinthat is adapted to allow a plurality of anions to pass through a cellmembrane in response to light. In some embodiments, the subjectcompositions include nucleic acids comprising nucleotide sequencesencoding the subject proteins, as well as additional components, such astranscriptional control elements (e.g., promoter sequences, such astissue-specific or cell type-specific promoter sequences, induciblepromoter sequences, and the like), trafficking sequences, signalsequences, endoplasmic reticulum export sequences, and the like. Each ofthese components is now further described in greater detail.

Engineered Anion Channel Proteins

As summarized above, aspects of the present disclosure includeengineered light-activated ion channel polypeptides that are adapted toallow one or more anions to pass through the plasma membrane of a targetcell when the polypeptide is illuminated with light of an activatingwavelength. The subject polypeptides are in some embodiments specific toa particular species of ion, meaning that the subject light-activatedpolypeptides only allow ions of a particular species to pass through themembrane of a cell. In some embodiments, the subject engineeredlight-activated anion channel polypeptides are specific to chloride ions(Cl⁻). In some embodiments, the subject engineered light-activated anionchannel polypeptides provide increased chloride conductivity and/orincreased chloride selectivity. In certain embodiments, the subjectengineered light-activated anion channel polypeptides provide strongerlight-induced inhibition of neuronal activity over a wider range ofconditions, such as high internal chloride concentrations.

In some embodiments, a subject light-activated polypeptide, whenexpressed on the membrane of a cell (e.g., a mammalian cell), and whenexposed to light of an activating wavelength, hyperpolarizes themembrane. In some embodiments, a subject light-activated polypeptideexhibits prolonged stability of photocurrents. In some embodiments, asubject light-activated polypeptide exhibits enhanced expression in cellmembranes and larger photocurrents in cultured neurons. In someembodiments, a subject light-activated polypeptide exhibits deceleratedchannel kinetics/decelerated channel closure. In some embodiments, asubject light-activated polypeptide conduct anions and inhibits theformation of action potentials in neurons for an extended period of time(e.g., from about 0.5 hours, up to about 0.75 hours, up to about 1 hour,up to about 1.25 hours, up to about 1.5 hours, up to about 1.75 hours,up to about 2 hours, up to about 2.25 hours, up to about 2.5 hours, upto about 2.75 hours, up to about 3 hours or more) after brief lightstimulations at lower light intensities.

In some embodiments, the subject engineered light-activated anionchannel polypeptides are activated by blue light. In some embodiments,the subject engineered light-activated anion channel polypeptides areactivated by green light. In some embodiments, the subject engineeredlight-activated anion channel polypeptides are activated by yellowlight. In some embodiments, the subject engineered light-activated anionchannel polypeptides are activated by orange light. In some embodiments,the subject engineered light-activated anion channel polypeptides areactivated by red light.

In some embodiments, the subject engineered light-activated anionchannel polypeptides are derived from a cation channel polypeptide thatcomprises an ion pore and a vestibule. In some embodiments, the aminoacid sequence of a cation channel polypeptide has been engineered tointroduce amino acid substitutions and/or sequence modifications thatchange the electrostatic potential of the polypeptide in the regionsurrounding the ion pore and the vestibule. In certain embodiments, theamino acid substitutions and/or sequence modifications are selected sothat the polarity of the cation channel polypeptide is reversed, therebyallowing the passage of anions through the pore instead of allowingcations through the pore. In some embodiments, the amino acidsubstitutions and/or sequence modifications are selected so that thesubject engineered anion channel polypeptides allow anions to passthrough the ion pore, while still maintaining appropriate proteinfolding, membrane expression, optical activation, and pore gating. Incertain embodiments, a subject polypeptide comprises an amino acidsequence that is at least 58% (e.g., at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%) identicalto SEQ ID NOS: 1, 23, 34 or 56 and functions as an anion channelprotein. In certain embodiments, a subject polypeptide comprises anamino acid sequence that is at least 60% (e.g., at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%) identical to SEQID NOS: 94, 116, 127 or 149, and functions as an anion channel protein.

Representative embodiments of the subject engineered light-activatedanion channel proteins are further described below.

Anion Channel Polypeptides Based on C1C2

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2. Theamino acid sequence of C1C2 is set forth in SEQ ID NO:78. In someembodiments, the amino acid sequence of the C1C2 protein has beenmodified by introducing one or more of the following mutations into theamino acid sequence: T98S, E129S, E140S, E162S, V156K, H173R, T285N,V281K and/or N297Q. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein C1C2 with all 9 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO: 1.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO: 1;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281Kand/or N297Q, relative to the amino acid sequence of C1C2 (SEQ IDNO:78). In some embodiments, a subject light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 1; and comprises T98S, E129S, E140S, E162S, and T285Nsubstitutions relative to the amino acid sequence of C1C2 (SEQ ID NO:78). This polypeptide is referred to herein as C1C2_5x. In someembodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO: 1;and comprises V156K, H173R, V281K, and N297Q substitutions relative tothe amino acid sequence of C1C2 (SEQ ID NO: 78). This polypeptide isreferred to herein as C1C2_4x.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO: 1;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162,K156, R173, N285, K281, and Q297, where the amino acid numbering is asset forth in SEQ ID NO: 1. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:1; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297, where the amino acidnumbering is as set forth in SEQ ID NO: 1. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:2;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162,K156, R173, N285, K281, and Q297, and comprises N195, where the aminoacid numbering is as set forth in SEQ ID NO:2. In some embodiments, asubject light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:2; and comprises S98,S129, S140, S162, K156, R173, N285, K281, and Q297, and comprises N195,where the amino acid numbering is as set forth in SEQ ID NO:2. In anyone of these embodiments, a subject anion channel polypeptide comprisesa membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)). In any one of these embodiments, a subject anion channelpolypeptide comprises an ER export signal (e.g., FCYENEV (SEQ IDNO:84)). In any one of these embodiments, a subject anion channelpolypeptide comprises both a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1C2 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:3; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, N246, K242, and Q258, where the amino acidnumbering is as set forth in SEQ ID NO:3. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:3; and comprises S59,S90, S101, S123, K117, R134, N246, K242, and Q258, where the amino acidnumbering is as set forth in SEQ ID NO:3. In some embodiments, a subjectlight-activated anion channel polypeptide comprises the amino acidsequence set forth in SEQ ID NO:3. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1C2 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:4; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, N246, K242, and Q258, and comprises N156, wherethe amino acid numbering is as set forth in SEQ ID NO:4. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:4; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258, and comprises N156, where the amino acid numbering is asset forth in SEQ ID NO:4. In some embodiments, a subject light-activatedanion channel polypeptide comprises the amino acid sequence set forth inSEQ ID NO:4. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the cysteine amino acid residue at position 167 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:5; and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281,and Q297; and comprises T167. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:5; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297; and comprises T167, wherethe amino acid numbering is as set forth in SEQ ID NO:5. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO:5. In some ofthese embodiments, the light-activated polypeptide exhibits prolongedstability of photocurrents. In some embodiments, the first 50 aminoacids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2,wherein the cysteine amino acid residue at position 167 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:6; and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281,and Q297; and comprises A167, where the amino acid numbering is as setforth in SEQ ID NO:6. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:6; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297; and comprises A167, wherethe amino acid numbering is as set forth in SEQ ID NO:6. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO:6. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2,wherein the cysteine amino acid residue at position 167 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:7; and comprises 1, 2, 3, 4, 5,6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281, andQ297; and comprises S167, where the amino acid numbering is as set forthin SEQ ID NO:7. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:7; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297; and comprises S167, wherethe amino acid numbering is as set forth in SEQ ID NO:7. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:7.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:8; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises T167, where the amino acid numbering is as set forth in SEQ IDNO:8. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:8; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises T167; and comprises A195, where theamino acid numbering is as set forth in SEQ ID NO:8. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:8.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:9; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises T167, where the amino acid numbering is as set forth in SEQ IDNO:9. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:9; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises T167; and comprises N195, where theamino acid numbering is as set forth in SEQ ID NO:9. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:9.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO: 10; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises A167, where the amino acid numbering is as set forth in SEQ IDNO: 10. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO: 10; and comprises S98, S129, S140, S162, K156,R173, N285, K281, and Q297; comprises A167; and comprises A195, wherethe amino acid numbering is as set forth in SEQ ID NO: 10. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO: 10.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO: 11; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises A167, where the amino acid numbering is as set forth in SEQ IDNO: 11. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO: 11; and comprises S98, S129, S140, S162, K156,R173, N285, K281, and Q297; comprises A167; and comprises N195, wherethe amino acid numbering is as set forth in SEQ ID NO: 11. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO: 11.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO: 12; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises S167, where the amino acid numbering is as set forth in SEQ IDNO: 12. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:12; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises S167; and comprises A195, where theamino acid numbering is as set forth in SEQ ID NO: 12. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO: 12.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO: 13; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises S167, where the amino acid numbering is as set forth in SEQ IDNO: 13. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO: 13; and comprises S98, S129, S140, S162, K156,R173, N285, K281, and Q297; comprises S167; and comprises N195, wherethe amino acid numbering is as set forth in SEQ ID NO: 13. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO: 13.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by analanine residue. In certain embodiments wherein the first 50 N-terminalamino acids of the protein are replaced by amino acids 1-11 from theprotein ChR2, the aspartate amino acid residue at position 156 (whichcorresponds to original position 195 of the C1C2 amino acid sequence setforth in SEQ ID NO:78) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by anasparagine residue. In certain embodiments wherein the first 50N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 195 of the C1C2 amino acidsequence set forth in SEQ ID NO:78) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 14; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128,where the amino acid numbering is as set forth in SEQ ID NO: 14. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 14; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises T128, where the amino acid numbering is asset forth in SEQ ID NO: 14. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 15; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128,where the amino acid numbering is as set forth in SEQ ID NO: 15. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 15; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises A128, where the amino acid numbering is asset forth in SEQ ID NO: 15. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 16; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128,where the amino acid numbering is as set forth in SEQ ID NO: 16. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 16; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises S128, where the amino acid numbering is asset forth in SEQ ID NO: 16. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 17; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and A156, where the amino acid numbering is as set forth in SEQ ID NO:17. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO: 17; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO: 17. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 18; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and N156, where the amino acid numbering is as set forth in SEQ ID NO:18. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO: 18; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO: 18. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 19; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and A156, where the amino acid numbering is as set forth in SEQ ID NO:19. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO: 19; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO: 19. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:20; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and N156, where the amino acid numbering is as set forth in SEQ IDNO:20. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:20; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:20. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:21; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and A156, where the amino acid numbering is as set forth in SEQ IDNO:21. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:21; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:21. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:22; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and N156, where the amino acid numbering is as set forth in SEQ IDNO:22. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:22; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:22. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinC1C2, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 1-22.

In some embodiments, the amino acid sequence of the C1C2 protein hasbeen modified by introducing one or more of the following mutations intothe amino acid sequence: T98S, E122N, E129Q, E140S, V156R, E162S, V281R,T285N, N297Q and E312S. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein C1C2 with all 10 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO: 94.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:94; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, V281R, T285N,N297Q and E312S, relative to the amino acid sequence of C1C2 (SEQ IDNO:78).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:94; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S, where the amino acid numbering isas set forth in SEQ ID NO:94. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO:94; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S, where the amino acid numbering isas set forth in SEQ ID NO:94. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:95; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S, and comprises 195N, where theamino acid numbering is as set forth in SEQ ID NO:95. In someembodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:95; andcomprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and 312S,and comprises 195N, where the amino acid numbering is as set forth inSEQ ID NO:95. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1C2 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:96;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: S59, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q and 273S, where the amino acid numbering isas set forth in SEQ ID NO:96. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO:96; and comprises S59, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q and 273S, where the amino acid numbering isas set forth in SEQ ID NO:96. In some embodiments, a subjectlight-activated anion channel polypeptide comprises the amino acidsequence set forth in SEQ ID NO:96. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1C2 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:97;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q and 273S, and comprises 156N, where theamino acid numbering is as set forth in SEQ ID NO:97. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:97;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S, and comprises 156N, where the amino acid numbering is as set forthin SEQ ID NO:97. In some embodiments, a subject light-activated anionchannel polypeptide comprises the amino acid sequence set forth in SEQID NO:97. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 (SEQID NO:78), wherein the cysteine amino acid residue at position 167 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO:98; and comprises 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; and comprises 167T. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:98; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; and comprises 167T, where theamino acid numbering is as set forth in SEQ ID NO:98. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO:98. In some ofthese embodiments, the light-activated polypeptide exhibits prolongedstability of photocurrents. In some embodiments, the first 50 aminoacids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2,wherein the cysteine amino acid residue at position 167 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO:99; and comprises 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; and comprises 167A, where the amino acid numbering is as set forthin SEQ ID NO:99. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:99; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; and comprises 167A, where theamino acid numbering is as set forth in SEQ ID NO:99. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO:99. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2,wherein the cysteine amino acid residue at position 167 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:100; and comprises 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; and comprises 167S, where the amino acid numbering is as set forthin SEQ ID NO: 100. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 100; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; and comprises 167S, where theamino acid numbering is as set forth in SEQ ID NO: 100. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:100. In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 101;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195C; and comprises167T, where the amino acid numbering is as set forth in SEQ ID NO: 101.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 101;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167T; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 101. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 101. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 102;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195N; and comprises167T, where the amino acid numbering is as set forth in SEQ ID NO: 102.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 102;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167T; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 102. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 102. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 103;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195C; and comprises167A, where the amino acid numbering is as set forth in SEQ ID NO: 103.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 103;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167A; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 103. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 103. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 104;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195N; and comprises167A, where the amino acid numbering is as set forth in SEQ ID NO: 104.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 104;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167A; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 104. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 104. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 105;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195C; and comprises167S, where the amino acid numbering is as set forth in SEQ ID NO: 105.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 105;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167S; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 105. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 105. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 106;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S; comprises 195N; and comprises167S, where the amino acid numbering is as set forth in SEQ ID NO: 106.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 106;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and312S; comprises 167S; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 106. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 106. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by analanine residue. In certain embodiments wherein the first 50 N-terminalamino acids of the protein are replaced by amino acids 1-11 from theprotein ChR2, the aspartate amino acid residue at position 156 (whichcorresponds to original position 195 of the C1C2 amino acid sequence setforth in SEQ ID NO:78) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1C2 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by anasparagine residue. In certain embodiments wherein the first 50N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 195 of the C1C2 amino acidsequence set forth in SEQ ID NO:78) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:107; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T, wherethe amino acid numbering is as set forth in SEQ ID NO: 107. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:107; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T, where the amino acid numbering is as set forthin SEQ ID NO: 107. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:108; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A, wherethe amino acid numbering is as set forth in SEQ ID NO: 108. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:108; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A, where the amino acid numbering is as set forthin SEQ ID NO: 108. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:109; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S, wherethe amino acid numbering is as set forth in SEQ ID NO: 109. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:109; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S, where the amino acid numbering is as set forthin SEQ ID NO: 109. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:110; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and 156Cwhere the amino acid numbering is as set forth in SEQ ID NO: 110. Insome embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:110; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 110. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:111; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and156N, where the amino acid numbering is as set forth in SEQ ID NO: 111.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:111; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 111. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:112; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and156C, where the amino acid numbering is as set forth in SEQ ID NO: 112.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:112; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 112. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 113; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of:59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and 273S; andcomprises 128A and 156N, where the amino acid numbering is as set forthin SEQ ID NO: 113. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 113; and comprises 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and 156N,where the amino acid numbering is as set forth in SEQ ID NO: 113. In anyone of these embodiments, a subject anion channel polypeptide comprisesa membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)). In any one of these embodiments, a subject anion channelpolypeptide comprises an ER export signal (e.g., FCYENEV (SEQ IDNO:84)). In any one of these embodiments, a subject anion channelpolypeptide comprises both a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:114; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156C, where the amino acid numbering is as set forth in SEQ ID NO: 114.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:114; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 114. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:115; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156N, where the amino acid numbering is as set forth in SEQ ID NO: 115.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:115; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 115. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinC1C2, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS:94-115.

Anion Channel Polypeptides Based on C1V1

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1. Theamino acid sequence of C1V1 is set forth in SEQ ID NO:80. In someembodiments, the amino acid sequence of the C1V1 protein has beenmodified by introducing one or more of the following mutations into theamino acid sequence: T98S, E129S, E140S, E162S, V156K, H173R, A285N,P281K and/or N297Q. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein C1V1 with all 9 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO:34.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:34;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281Kand/or N297Q, relative to the amino acid sequence of C1V1 (SEQ IDNO:80).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:34;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162,K156, R173, N285, K281, and Q297, where the amino acid numbering is asset forth in SEQ ID NO:34. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:34; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297, where the amino acidnumbering is as set forth in SEQ ID NO:34. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:35;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162,K156, R173, N285, K281, and Q297, and comprises N195, where the aminoacid numbering is as set forth in SEQ ID NO:35. In some embodiments, asubject light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:35; and comprises S98,S129, S140, S162, K156, R173, N285, K281, and Q297, and comprises N195,where the amino acid numbering is as set forth in SEQ ID NO:35. In anyone of these embodiments, a subject anion channel polypeptide comprisesa membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)). In any one of these embodiments, a subject anion channelpolypeptide comprises an ER export signal (e.g., FCYENEV (SEQ IDNO:84)). In any one of these embodiments, a subject anion channelpolypeptide comprises both a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1V1 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:36; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258, where the amino acidnumbering is as set forth in SEQ ID NO:36. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesan amino acid sequence having at least 58%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:36; andcomprises S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherethe amino acid numbering is as set forth in SEQ ID NO:36. In someembodiments, a subject light-activated anion channel polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:36. In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1V1 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:37; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258, and comprises N156,where the amino acid numbering is as set forth in SEQ ID NO:37. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:37; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258, and comprises N156, where the amino acid numbering is asset forth in SEQ ID NO:37. In some embodiments, a subjectlight-activated anion channel polypeptide comprises the amino acidsequence set forth in SEQ ID NO:37. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the cysteine amino acid residue at position 167 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:38; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285,K281, and Q297; and comprises T167. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:38; and comprises S98,S129, S140, S162, K156, R173, N285, K281, and Q297; and comprises T167,where the amino acid numbering is as set forth in SEQ ID NO:38. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO:38. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1,wherein the cysteine amino acid residue at position 167 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:39; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285,K281, and Q297; and comprises A167, where the amino acid numbering is asset forth in SEQ ID NO:39. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:39; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297; and comprises A167, wherethe amino acid numbering is as set forth in SEQ ID NO:39. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO:39. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1,wherein the cysteine amino acid residue at position 167 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:40; and comprises 1, 2, 3, 4, 5,6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281, andQ297; and comprises S167, where the amino acid numbering is as set forthin SEQ ID NO:40. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:40; and comprises S98, S129,S140, S162, K156, R173, N285, K281, and Q297; and comprises S167, wherethe amino acid numbering is as set forth in SEQ ID NO:40. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:40.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:41; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises T167, where the amino acid numbering is as set forth in SEQ IDNO:41. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:41; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises T167; and comprises A195, where theamino acid numbering is as set forth in SEQ ID NO:41. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:41.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:42; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises T167, where the amino acid numbering is as set forth in SEQ IDNO:42. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:42; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises T167; and comprises N195, where theamino acid numbering is as set forth in SEQ ID NO:42. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:42.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:43; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises A167, where the amino acid numbering is as set forth in SEQ IDNO:43. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:43; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises A167; and comprises A195, where theamino acid numbering is as set forth in SEQ ID NO:43. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:43.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:44; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises A167, where the amino acid numbering is as set forth in SEQ IDNO:44. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:44; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises A167; and comprises N195, where theamino acid numbering is as set forth in SEQ ID NO:44. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:44.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:45; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises A195; andcomprises S167, where the amino acid numbering is as set forth in SEQ IDNO:45. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:45; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises S167; and comprises A195, where theamino acid numbering is as set forth in SEQ ID NO:45. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:45.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:46; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,S140, S162, K156, R173, N285, K281, and Q297; comprises N195; andcomprises S167, where the amino acid numbering is as set forth in SEQ IDNO:46. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:46; and comprises S98, S129, S140, S162, K156, R173,N285, K281, and Q297; comprises S167; and comprises N195, where theamino acid numbering is as set forth in SEQ ID NO:46. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:46.In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by analanine residue. In certain embodiments wherein the first 50 N-terminalamino acids of the protein are replaced by amino acids 1-11 from theprotein ChR2, the aspartate amino acid residue at position 156 (whichcorresponds to original position 195 of the C1V1 amino acid sequence setforth in SEQ ID NO:80) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by anasparagine residue. In certain embodiments wherein the first 50N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 195 of the C1V1 amino acidsequence set forth in SEQ ID NO:80) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:47; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128,where the amino acid numbering is as set forth in SEQ ID NO:47. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:47; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises T128, where the amino acid numbering is asset forth in SEQ ID NO:47. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:48; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128,where the amino acid numbering is as set forth in SEQ ID NO:48. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:48; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises A128, where the amino acid numbering is asset forth in SEQ ID NO:48. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:49; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128,where the amino acid numbering is as set forth in SEQ ID NO:49. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:49; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises S128, where the amino acid numbering is asset forth in SEQ ID NO:49. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:50; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and A156, where the amino acid numbering is as set forth in SEQ IDNO:50. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:50; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:50. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:51; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and N156, where the amino acid numbering is as set forth in SEQ IDNO:51. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:51; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:51. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:52; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and A156, where the amino acid numbering is as set forth in SEQ IDNO:52. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:52; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:52. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:53; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and N156, where the amino acid numbering is as set forth in SEQ IDNO:53. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:53; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:53. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:54; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and A156, where the amino acid numbering is as set forth in SEQ IDNO:54. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:54; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:54. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:55; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and N156, where the amino acid numbering is as set forth in SEQ IDNO:55. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:55; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:55. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinC1V1, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 34-55.

In some embodiments, the amino acid sequence of the C1V1 protein hasbeen modified by introducing one or more of the following mutations intothe amino acid sequence: T98S, E122N, E129Q, E140S, V156R, E162S, P281R,A285N, N297Q and/or E312S. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein C1V1 with all 10 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO: 127.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 127; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 127; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S, where the amino acid numbering isas set forth in SEQ ID NO: 127. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 127; and comprises 98S, 122N, 129Q,140S, 156R, 162S, 281R, 285N, 297Q and 312S, where the amino acidnumbering is as set forth in SEQ ID NO: 127. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 128; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q and 312S, and comprises 195N, where theamino acid numbering is as set forth in SEQ ID NO: 128. In someembodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 128; andcomprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q and 312S,and comprises 195N, where the amino acid numbering is as set forth inSEQ ID NO: 128. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1V1 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:129; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S, where the amino acidnumbering is as set forth in SEQ ID NO: 129. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesan amino acid sequence having at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO: 129; and comprises 59S, 83N,90Q, 101S, 117R, 123S, 242R, 246N, 258Q and 273S, where the amino acidnumbering is as set forth in SEQ ID NO: 129. In some embodiments, asubject light-activated anion channel polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 129. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the amino acid sequence has been modified byreplacing the first 50 N-terminal amino acids of C1V1 with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:130; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S, and comprises 156N, wherethe amino acid numbering is as set forth in SEQ ID NO: 130. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO: 130; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R,246N, 258Q and 273S, and comprises 156N, where the amino acid numberingis as set forth in SEQ ID NO: 130. In some embodiments, a subjectlight-activated anion channel polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 130. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 (SEQID NO:80), wherein the cysteine amino acid residue at position 167 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO:131; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q,and 312S; and comprises 167T. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 131; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; and comprises 167T, where theamino acid numbering is as set forth in SEQ ID NO: 131. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO: 131. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1,wherein the cysteine amino acid residue at position 167 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO: 132; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q,and 312S; and comprises 167A, where the amino acid numbering is as setforth in SEQ ID NO: 132. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 132; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; and comprises 167A, where theamino acid numbering is as set forth in SEQ ID NO: 132. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO: 132. In someembodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1,wherein the cysteine amino acid residue at position 167 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:133; and comprises 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 of: 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; and comprises 167S, where the amino acid numbering is as set forthin SEQ ID NO: 133. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 133; and comprises 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; and comprises 167S, where theamino acid numbering is as set forth in SEQ ID NO: 133. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:133. In some embodiments, the first 50 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 134;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195C and comprises167T, where the amino acid numbering is as set forth in SEQ ID NO: 134.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 134;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167T; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 134. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 134. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 135;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195N; and comprises167T, where the amino acid numbering is as set forth in SEQ ID NO: 135.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 135;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167T; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 135. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 135. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 136;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195C and comprises167A, where the amino acid numbering is as set forth in SEQ ID NO: 136.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 136;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167A; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 136. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 136. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 137;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195N; and comprises167A, where the amino acid numbering is as set forth in SEQ ID NO: 137.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 137;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167A; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 137. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 137. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 138;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195C; and comprises167S, where the amino acid numbering is as set forth in SEQ ID NO: 138.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 138;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167S; and comprises 195C, where the amino acid numberingis as set forth in SEQ ID NO: 138. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 138. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 139;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 98S, 122N, 129Q, 140S,156R, 162S, 281R, 285N, 297Q, and 312S; comprises 195N; and comprises167S, where the amino acid numbering is as set forth in SEQ ID NO: 139.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 139;and comprises 98S, 122N, 129Q, 140S, 156R, 162S, 281R, 285N, 297Q, and312S; comprises 167S; and comprises 195N, where the amino acid numberingis as set forth in SEQ ID NO: 139. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 139. In some embodiments, the first50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by analanine residue. In certain embodiments wherein the first 50 N-terminalamino acids of the protein are replaced by amino acids 1-11 from theprotein ChR2, the aspartate amino acid residue at position 156 (whichcorresponds to original position 195 of the C1V1 amino acid sequence setforth in SEQ ID NO:80) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein C1V1 withone or more of the modifications described above, wherein the aspartateamino acid residue at original position 195 has been replaced by anasparagine residue. In certain embodiments wherein the first 50N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 195 of the C1V1 amino acidsequence set forth in SEQ ID NO:80) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:140; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T, wherethe amino acid numbering is as set forth in SEQ ID NO: 140. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:140; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T, where the amino acid numbering is as set forthin SEQ ID NO: 140. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:141; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A, wherethe amino acid numbering is as set forth in SEQ ID NO: 141. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:141; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A, where the amino acid numbering is as set forthin SEQ ID NO: 141. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:142; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S, wherethe amino acid numbering is as set forth in SEQ ID NO: 142. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:142; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S, where the amino acid numbering is as set forthin SEQ ID NO: 142. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:143; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and156C, where the amino acid numbering is as set forth in SEQ ID NO: 143.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:143; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 143. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:144; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and156N, where the amino acid numbering is as set forth in SEQ ID NO: 144.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:144; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 144. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:145; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and156C, where the amino acid numbering is as set forth in SEQ ID NO: 145.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:145; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 145. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:146; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and156N, where the amino acid numbering is as set forth in SEQ ID NO: 146.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:146; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 146. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:147; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156C, where the amino acid numbering is as set forth in SEQ ID NO: 147.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:147; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 147. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:148; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156N, where the amino acid numbering is as set forth in SEQ ID NO: 148.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:148; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 148. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinC1V1, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 127-148.

Anion Channel Polypeptides based on ReaChR

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR.The amino acid sequence of ReaChR is set forth in SEQ ID NO: 81. In someembodiments, the amino acid sequence of the ReaChR protein has beenmodified by introducing one or more of the following mutations into theamino acid sequence: T99S, E130S, E141S, E163S, V157K, H174R, A286N,P282K and/or N298Q. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein ReaChR with all 9 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO:56.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:56;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from T99S, E130S, E141S, E163S, V157K, H174R, A286N, P282Kand/or N298Q, relative to the amino acid sequence of ReaChR (SEQ IDNO:81).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:56;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163,K157, R174, N286, K281, and Q298, where the amino acid numbering is asset forth in SEQ ID NO:56. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:56; and comprises S99, S130,S141, S163, K157, R174, N286, K281, and Q298, where the amino acidnumbering is as set forth in SEQ ID NO:56. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:57;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163,K157, R174, N286, K281, and Q298, and comprises N196, where the aminoacid numbering is as set forth in SEQ ID NO:57. In some embodiments, asubject light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:57; and comprises S99,S130, S141, S163, K157, R174, N286, K281, and Q298, and comprises N196,where the amino acid numbering is as set forth in SEQ ID NO:57. In anyone of these embodiments, a subject anion channel polypeptide comprisesa membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)). In any one of these embodiments, a subject anion channelpolypeptide comprises an ER export signal (e.g., FCYENEV (SEQ IDNO:84)). In any one of these embodiments, a subject anion channelpolypeptide comprises both a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO:81), wherein the amino acid sequence has been modified byreplacing the first 51 N-terminal amino acids of ReaChR with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:58; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258, where the amino acidnumbering is as set forth in SEQ ID NO:58. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesan amino acid sequence having at least 58%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:58; andcomprises S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherethe amino acid numbering is as set forth in SEQ ID NO:58. In someembodiments, a subject light-activated anion channel polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:58. In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO:81), wherein the amino acid sequence has been modified byreplacing the first 51 N-terminal amino acids of ReaChR with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:59; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258, and comprises N156,where the amino acid numbering is as set forth in SEQ ID NO:59. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:59; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258, and comprises N156, where the amino acid numbering is asset forth in SEQ ID NO:59. In some embodiments, a subjectlight-activated anion channel polypeptide comprises the amino acidsequence set forth in SEQ ID NO:59. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO:81), wherein the cysteine amino acid residue at position 168has been replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:60; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163, K157, R174, N286,K281, and Q298; and comprises T168. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:60; and comprises S99,S130, S141, S163, K157, R174, N286, K281, and Q298; and comprises T168,where the amino acid numbering is as set forth in SEQ ID NO:60. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO:60. In someembodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR,wherein the cysteine amino acid residue at position 168 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:61; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163, K157, R174, N286,K281, and Q298; and comprises A168, where the amino acid numbering is asset forth in SEQ ID NO:61. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:61; and comprises S99, S130,S141, S163, K157, R174, N286, K281, and Q298; and comprises A168, wherethe amino acid numbering is as set forth in SEQ ID NO:61. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO:61. In someembodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR,wherein the cysteine amino acid residue at position 168 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:62; and comprises 1, 2, 3, 4, 5,6, 7, 8, or 9 of: S99, S130, S141, S163, K157, R174, N286, K281, andQ298; and comprises S168, where the amino acid numbering is as set forthin SEQ ID NO:62. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:62; and comprises S99, S130,S141, S163, K157, R174, N286, K281, and Q298; and comprises S168, wherethe amino acid numbering is as set forth in SEQ ID NO:62. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:62.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:63; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises A196; andcomprises T168, where the amino acid numbering is as set forth in SEQ IDNO:63. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:63; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises T168; and comprises A196, where theamino acid numbering is as set forth in SEQ ID NO:63. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:63.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:64; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises N196; andcomprises T168, where the amino acid numbering is as set forth in SEQ IDNO:64. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:64; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises T168; and comprises N196, where theamino acid numbering is as set forth in SEQ ID NO:64. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:64.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:65; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises A196; andcomprises A168, where the amino acid numbering is as set forth in SEQ IDNO:65. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:65; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises A168; and comprises A196, where theamino acid numbering is as set forth in SEQ ID NO:65. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:65.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:66; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises N196; andcomprises A168, where the amino acid numbering is as set forth in SEQ IDNO:66. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:66; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises A168; and comprises N196, where theamino acid numbering is as set forth in SEQ ID NO:66. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:66.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:67; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises A196; andcomprises S168, where the amino acid numbering is as set forth in SEQ IDNO:67. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:67; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises S168; and comprises A196, where theamino acid numbering is as set forth in SEQ ID NO:67. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:67.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:68; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130,S141, S163, K157, R174, N286, K281, and Q298; comprises N196; andcomprises S168, where the amino acid numbering is as set forth in SEQ IDNO:68. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:68; and comprises S99, S130, S141, S163, K157, R174,N286, K281, and Q298; comprises S168; and comprises N196, where theamino acid numbering is as set forth in SEQ ID NO:68. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:68.In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChRwith one or more of the modifications described above, wherein theaspartate amino acid residue at original position 196 has been replacedby an alanine residue. In certain embodiments wherein the first 51N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 196 of the ReaChR amino acidsequence set forth in SEQ ID NO:81) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChRwith one or more of the modifications described above, wherein theaspartate amino acid residue at original position 196 has been replacedby an asparagine residue. In certain embodiments wherein the first 51N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 196 of the ReaChR amino acidsequence set forth in SEQ ID NO:81) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:69; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128,where the amino acid numbering is as set forth in SEQ ID NO:69. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:69; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises T128, where the amino acid numbering is asset forth in SEQ ID NO:69. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:70; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128,where the amino acid numbering is as set forth in SEQ ID NO:70. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:70; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises A128, where the amino acid numbering is asset forth in SEQ ID NO:70. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:71; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128,where the amino acid numbering is as set forth in SEQ ID NO:71. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:71; and comprises S59, S90, S101, S123, K117, R134, N246,K242, and Q258; and comprises S128, where the amino acid numbering is asset forth in SEQ ID NO:71. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:72; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and A156, where the amino acid numbering is as set forth in SEQ IDNO:72. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:72; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:72. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:73; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises T128and N156, where the amino acid numbering is as set forth in SEQ IDNO:73. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:73; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises T128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:73. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:74; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and A156, where the amino acid numbering is as set forth in SEQ IDNO:74. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:74; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:74. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:75; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises A128and N156, where the amino acid numbering is as set forth in SEQ IDNO:75. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:75; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises A128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:75. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:76; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and A156, where the amino acid numbering is as set forth in SEQ IDNO:76. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:76; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and A156, where the aminoacid numbering is as set forth in SEQ ID NO:76. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:77; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59,S90, S101, S123, K117, R134, N246, K242, and Q258; and comprises S128and N156, where the amino acid numbering is as set forth in SEQ IDNO:77. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:77; and comprises S59, S90, S101, S123, K117,R134, N246, K242, and Q258; and comprises S128 and N156, where the aminoacid numbering is as set forth in SEQ ID NO:77. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinReaChR, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 56-77.

In some embodiments, the amino acid sequence of the ReaChR protein hasbeen modified by introducing one or more of the following mutations intothe amino acid sequence: T99S, E123N, E130Q, E141S, V157R, E163S, P282R,A286N, N298Q and/or E313S. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein ReaChR with all 10 of the above-listed aminoacid substitutions, such that the amino acid sequence of the polypeptideis provided in SEQ ID NO: 149.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 149; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T99S, E123N, E130Q, E141S, V157R, E163S, P282R, A286N,N298Q and/or E313S, relative to the amino acid sequence of ReaChR (SEQID NO:81).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 149; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, E163S, 282R, 286N, 298Q and 313S, where the amino acid numberingis as set forth in SEQ ID NO: 149. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 149; and comprises 99S, 123N, 130Q,141S, 157R, E163S, 282R, 286N, 298Q and 313S, where the amino acidnumbering is as set forth in SEQ ID NO: 149. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 150; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, E163S, 282R, 286N, 298Q and 313S, and comprises 196N, where theamino acid numbering is as set forth in SEQ ID NO: 150. In someembodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:150; andcomprises 99S, 123N, 130Q, 141S, 157R, E163S, 282R, 286N, 298Q and 313S,and comprises 196N, where the amino acid numbering is as set forth inSEQ ID NO: 150. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO:81), wherein the amino acid sequence has been modified byreplacing the first 51 N-terminal amino acids of ReaChR with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:151; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S, where the amino acidnumbering is as set forth in SEQ ID NO: 151. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesan amino acid sequence having at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO: 151; and comprises 59S, 83N,90Q, 101S, 117R, 123S, 242R, 246N, 258Q and 273S, where the amino acidnumbering is as set forth in SEQ ID NO: 151. In some embodiments, asubject light-activated anion channel polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 151. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO: 81), wherein the amino acid sequence has been modified byreplacing the first 51 N-terminal amino acids of ReaChR with amino acids1-11 from the protein ChR2 (MDYGGALSAVG) (SEQ ID NO:82). In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:152; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S, and comprises 156N, wherethe amino acid numbering is as set forth in SEQ ID NO: 152. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:152;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S, and comprises 156N, where the amino acid numbering is as set forthin SEQ ID NO: 152. In some embodiments, a subject light-activated anionchannel polypeptide comprises the amino acid sequence set forth in SEQID NO: 152. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR(SEQ ID NO:81), wherein the cysteine amino acid residue at position 168has been replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO: 153; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Qand 313S; and comprises 168T. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 153; and comprises 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; and comprises 168T, where theamino acid numbering is as set forth in SEQ ID NO: 153. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO: 153. In someembodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR,wherein the cysteine amino acid residue at position 168 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO: 154; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Qand 313S; and comprises 168A, where the amino acid numbering is as setforth in SEQ ID NO: 154. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 154; and comprises 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; and comprises 168A, where theamino acid numbering is as set forth in SEQ ID NO: 154. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO: 154. In someembodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQID NO:82). In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChR,wherein the cysteine amino acid residue at position 168 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:155; and comprises 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 of: 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; and comprises 168S, where the amino acid numbering is as set forthin SEQ ID NO: 155. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 155; and comprises 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; and comprises 168S, where theamino acid numbering is as set forth in SEQ ID NO: 155. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:155. In some embodiments, the first 51 amino acids are replaced withMDYGGALSAVG (SEQ ID NO:82). In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 156;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196C; and comprises168T, where the amino acid numbering is as set forth in SEQ ID NO: 156.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 156;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168T; and comprises 196C, where the amino acid numberingis as set forth in SEQ ID NO: 156. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 156. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 157;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196N; and comprises168T, where the amino acid numbering is as set forth in SEQ ID NO: 157.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 157;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168T; and comprises 196N, where the amino acid numberingis as set forth in SEQ ID NO: 157. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 157. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 158;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196C; and comprises168A, where the amino acid numbering is as set forth in SEQ ID NO: 158.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 158;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168A; and comprises 196C, where the amino acid numberingis as set forth in SEQ ID NO: 158. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 158. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 159;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196N; and comprises168A, where the amino acid numbering is as set forth in SEQ ID NO: 159.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 159;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168A; and comprises 196N, where the amino acid numberingis as set forth in SEQ ID NO: 159. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 159. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 160;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196C; and comprises168S, where the amino acid numbering is as set forth in SEQ ID NO: 160.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 160;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168S; and comprises 196C, where the amino acid numberingis as set forth in SEQ ID NO: 160. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 160. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 161;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 99S, 123N, 130Q, 141S,157R, 163S, 282R, 286N, 298Q and 313S; comprises 196N; and comprises168S, where the amino acid numbering is as set forth in SEQ ID NO: 161.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 161;and comprises 99S, 123N, 130Q, 141S, 157R, 163S, 282R, 286N, 298Q and313S; comprises 168S; and comprises 196N, where the amino acid numberingis as set forth in SEQ ID NO: 161. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 161. In some embodiments, the first51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:82). In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChRwith one or more of the modifications described above, wherein theaspartate amino acid residue at original position 196 has been replacedby an alanine residue. In certain embodiments wherein the first 51N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 196 of the ReaChR amino acidsequence set forth in SEQ ID NO:81) is replaced by an alanine residue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ReaChRwith one or more of the modifications described above, wherein theaspartate amino acid residue at original position 196 has been replacedby an asparagine residue. In certain embodiments wherein the first 51N-terminal amino acids of the protein are replaced by amino acids 1-11from the protein ChR2, the aspartate amino acid residue at position 156(which corresponds to original position 196 of the ReaChR amino acidsequence set forth in SEQ ID NO:81) is replaced by an asparagineresidue.

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:162; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T, wherethe amino acid numbering is as set forth in SEQ ID NO: 162. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:162; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T, where the amino acid numbering is as set forthin SEQ ID NO: 162. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:163; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A, wherethe amino acid numbering is as set forth in SEQ ID NO: 163. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:163; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A, where the amino acid numbering is as set forthin SEQ ID NO: 163. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:164; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S, wherethe amino acid numbering is as set forth in SEQ ID NO: 164. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:164; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S, where the amino acid numbering is as set forthin SEQ ID NO: 164. In any one of these embodiments, a subject anionchannel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:165; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and156C, where the amino acid numbering is as set forth in SEQ ID NO: 165.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:165; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 165. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:166; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128T and156N, where the amino acid numbering is as set forth in SEQ ID NO: 166.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:166; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128T and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 166. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:167; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and156C, where the amino acid numbering is as set forth in SEQ ID NO: 167.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:167; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 167. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:168; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128A and156N, where the amino acid numbering is as set forth in SEQ ID NO: 168.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:168; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128A and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 168. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:169; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156C, where the amino acid numbering is as set forth in SEQ ID NO: 169.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:169; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156C, where the amino acid numbering is asset forth in SEQ ID NO: 169. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:170; and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q and 273S; and comprises 128S and156N, where the amino acid numbering is as set forth in SEQ ID NO: 170.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:170; and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q and273S; and comprises 128S and 156N, where the amino acid numbering is asset forth in SEQ ID NO: 170. In any one of these embodiments, a subjectanion channel polypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinReaChR, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 149-170.

Anion Channel Proteins Based on ChR2

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2. Theamino acid sequence of ChR2 is set forth in SEQ ID NO:79. In someembodiments, the amino acid sequence of the ChR2 protein has beenmodified by introducing one or more of the following mutations into theamino acid sequence: A59S, E90S, E101S, E123S, Q117K, H134R, V242K,T246N and/or N258Q. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein ChR2 with all 9 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO:23.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:23;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutionsselected from A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246Nand/or N258Q, relative to the amino acid sequence of ChR2 (SEQ IDNO:79).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:23;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,K117, R134, K242, N246 and Q258, where the amino acid numbering is asset forth in SEQ ID NO:23. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:23; and comprises S59, S90,S101, S123, K117, R134, K242, N246 and Q258, where the amino acidnumbering is as set forth in SEQ ID NO:23. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 58%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:24;and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,K117, R134, K242, N246 and Q258, and comprises N156, where the aminoacid numbering is as set forth in SEQ ID NO:24. In some embodiments, asubject light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:24; and comprises S59,S90, S101, S123, K117, R134, K242, N246 and Q258, and comprises N156,where the amino acid numbering is as set forth in SEQ ID NO:24. In anyone of these embodiments, a subject anion channel polypeptide comprisesa membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)). In any one of these embodiments, a subject anion channelpolypeptide comprises an ER export signal (e.g., FCYENEV (SEQ IDNO:84)). In any one of these embodiments, a subject anion channelpolypeptide comprises both a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2 (SEQID NO:79), wherein the cysteine amino acid residue at position 128 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:25; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134, K242, N246and Q258; and comprises T128. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:25; and comprises S59, S90,S101, S123, K117, R134, K242, N246 and Q258; and comprises T128, wherethe amino acid numbering is as set forth in SEQ ID NO:25. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO:25. In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2,wherein the cysteine amino acid residue at position 128 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 58%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, amino acid sequence identityto the amino acid sequence set forth SEQ ID NO:26; and comprises 1, 2,3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134, K242, N246and Q258; and comprises A128, where the amino acid numbering is as setforth in SEQ ID NO:26. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:26; and comprises S59, S90,S101, S123, K117, R134, K242, N246 and Q258; and comprises A128, wherethe amino acid numbering is as set forth in SEQ ID NO:26. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO:26. In any one ofthese embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2,wherein the cysteine amino acid residue at position 128 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 58%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to theamino acid sequence set forth SEQ ID NO:27; and comprises 1, 2, 3, 4, 5,6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134, K242, N246 and Q258;and comprises S128, where the amino acid numbering is as set forth inSEQ ID NO:27. In some embodiments, a subject engineered light-activatedanion channel polypeptide comprises an amino acid sequence having atleast 58%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:27; and comprises S59, S90, S101, S123,K117, R134, K242, N246 and Q258; and comprises S128, where the aminoacid numbering is as set forth in SEQ ID NO:27. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:27. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:28; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises A156; andcomprises T128, where the amino acid numbering is as set forth in SEQ IDNO:28. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:28; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises T128; and comprises A156, where the aminoacid numbering is as set forth in SEQ ID NO:28. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:28. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:29; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises N156; andcomprises T128, where the amino acid numbering is as set forth in SEQ IDNO:29. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:29; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises T128; and comprises N156, where the aminoacid numbering is as set forth in SEQ ID NO:29. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:29. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:30; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises A128; andcomprises A156, where the amino acid numbering is as set forth in SEQ IDNO:30. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:30; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises A128; and comprises A156, where the aminoacid numbering is as set forth in SEQ ID NO:30. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:30. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:31; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises A128; andcomprises N156, where the amino acid numbering is as set forth in SEQ IDNO:31. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:31; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises A128; and comprises N156, where the aminoacid numbering is as set forth in SEQ ID NO:31. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:31. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:32; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises A156; andcomprises S128, where the amino acid numbering is as set forth in SEQ IDNO:32. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:32; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises S128; and comprises A156, where the aminoacid numbering is as set forth in SEQ ID NO:32. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:32. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 58%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to the amino acid sequence set forthSEQ ID NO:33; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90,S101, S123, K117, R134, K242, N246 and Q258; comprises N156; andcomprises S128, where the amino acid numbering is as set forth in SEQ IDNO:33. In some embodiments, a subject engineered light-activated anionchannel polypeptide comprises an amino acid sequence having at least58%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, amino acid sequence identity to the amino acid sequenceset forth SEQ ID NO:33; and comprises S59, S90, S101, S123, K117, R134,K242, N246 and Q258; comprises S128; and comprises N156, where the aminoacid numbering is as set forth in SEQ ID NO:33. In some embodiments, asubject engineered light-activated anion channel polypeptide comprisesthe amino acid sequence provided in SEQ ID NO:33. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinChR2, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS:23-33.

In some embodiments, the amino acid sequence of the ChR2 protein hasbeen modified by introducing one or more of the following mutations intothe amino acid sequence: A59S, E83N, E90Q, E101S, Q117R, E123S, V242R,T246N, N258Q, and/or E273S. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises the amino acidsequence of the protein ChR2 with all 10 of the above-listed amino acidsubstitutions, such that the amino acid sequence of the polypeptide isprovided in SEQ ID NO: 116.

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 116; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from A59S, E83N, E90Q, E101S, Q117R, E123S, V242R, T246N,N258Q, and/or E273S, relative to the amino acid sequence of ChR2 (SEQ IDNO:79).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 116; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S, 117R,123S, 242R, 246N, 258Q, and 273S, where the amino acid numbering is asset forth in SEQ ID NO: 116. In some embodiments, a subjectlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 116; and comprises 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S, where the amino acid numberingis as set forth in SEQ ID NO: 116. In any one of these embodiments, asubject anion channel polypeptide comprises a membrane traffickingsignal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of theseembodiments, a subject anion channel polypeptide comprises an ER exportsignal (e.g., FCYENEV (SEQ ID NO:84)). In any one of these embodiments,a subject anion channel polypeptide comprises both a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ERexport signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject light-activated anion channel polypeptidecomprises an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 117; andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S, 117R,123S, 242R, 246N, 258Q, and 273S, and comprises 156N, where the aminoacid numbering is as set forth in SEQ ID NO: 117. In some embodiments, asubject light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO:117; and comprises 59S, 83N, 90Q,101S, 117R, 123S, 242R, 246N, 258Q, and 273S, and comprises 156N, wherethe amino acid numbering is as set forth in SEQ ID NO: 117. In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2 (SEQID NO:79), wherein the cysteine amino acid residue at position 128 hasbeen replaced by a threonine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO: 118; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; and comprises 128T. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 118; and comprises 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; and comprises 128T, where theamino acid numbering is as set forth in SEQ ID NO: 118. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino acid sequence provided in SEQ ID NO: 118. In any oneof these embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2,wherein the cysteine amino acid residue at position 128 has beenreplaced by an alanine residue. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises an aminoacid sequence having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to the aminoacid sequence set forth SEQ ID NO: 119; and comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; and comprises 128A, where the amino acid numbering is as set forthin SEQ ID NO: 119. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 119; and comprises 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; and comprises 128A, where theamino acid numbering is as set forth in SEQ ID NO: 119. In someembodiments, the engineered light-activated anion channel polypeptidecomprises the amino sequence provided in SEQ ID NO: 119. In any one ofthese embodiments, a subject anion channel polypeptide comprises amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).In any one of these embodiments, a subject anion channel polypeptidecomprises an ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any oneof these embodiments, a subject anion channel polypeptide comprises botha membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ IDNO:83)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide is based on the amino acid sequence of the protein ChR2,wherein the cysteine amino acid residue at position 128 has beenreplaced by a serine residue. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO:120; and comprises 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 of: 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and 273S;and comprises 128S, where the amino acid numbering is as set forth inSEQ ID NO: 120. In some embodiments, a subject engineeredlight-activated anion channel polypeptide comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the amino acidsequence set forth SEQ ID NO: 120; and comprises 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; and comprises 128S, where theamino acid numbering is as set forth in SEQ ID NO: 120. In someembodiments, a subject engineered light-activated anion channelpolypeptide comprises the amino acid sequence provided in SEQ ID NO:120. In any one of these embodiments, a subject anion channelpolypeptide comprises a membrane trafficking signal (e.g.,KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In any one of these embodiments, asubject anion channel polypeptide comprises an ER export signal (e.g.,FCYENEV (SEQ ID NO:84)). In any one of these embodiments, a subjectanion channel polypeptide comprises both a membrane trafficking signal(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)) and an ER export signal(e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 121;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 156C; and comprises128T, where the amino acid numbering is as set forth in SEQ ID NO: 121.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 121;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128T; and comprises 156C, where the amino acid numberingis as set forth in SEQ ID NO: 121. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 121. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 122;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 156N; and comprises128T, where the amino acid numbering is as set forth in SEQ ID NO: 122.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 122;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128T; and comprises 156N, where the amino acid numberingis as set forth in SEQ ID NO: 122. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 122. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 123;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 128A; and comprises156C, where the amino acid numbering is as set forth in SEQ ID NO: 123.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 123;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128A; and comprises 156C, where the amino acid numberingis as set forth in SEQ ID NO: 123. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 123. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 124;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 128A; and comprises156N, where the amino acid numbering is as set forth in SEQ ID NO: 124.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 124;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128A; and comprises 156N, where the amino acid numberingis as set forth in SEQ ID NO: 124. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 124. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 125;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 156C; and comprises128S, where the amino acid numbering is as set forth in SEQ ID NO: 125.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 125;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128S; and comprises 156C, where the amino acid numberingis as set forth in SEQ ID NO: 125. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 125. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 126;and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of: 59S, 83N, 90Q, 101S,117R, 123S, 242R, 246N, 258Q, and 273S; comprises 156N; and comprises128S, where the amino acid numbering is as set forth in SEQ ID NO: 126.In some embodiments, a subject engineered light-activated anion channelpolypeptide comprises an amino acid sequence having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence set forth SEQ ID NO: 126;and comprises 59S, 83N, 90Q, 101S, 117R, 123S, 242R, 246N, 258Q, and273S; comprises 128S; and comprises 156N, where the amino acid numberingis as set forth in SEQ ID NO: 126. In some embodiments, a subjectengineered light-activated anion channel polypeptide comprises the aminoacid sequence provided in SEQ ID NO: 126. In any one of theseembodiments, a subject anion channel polypeptide comprises a membranetrafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)). In anyone of these embodiments, a subject anion channel polypeptide comprisesan ER export signal (e.g., FCYENEV (SEQ ID NO:84)). In any one of theseembodiments, a subject anion channel polypeptide comprises both amembrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83))and an ER export signal (e.g., FCYENEV (SEQ ID NO:84)).

In certain embodiments, a subject engineered light-activated anionchannel polypeptide is based on the amino acid sequence of the proteinChR2, wherein the amino acid sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceprovided in one of SEQ ID NOS: 116-126.

Enhanced Intracellular Transport Amino Acid Motifs

The present disclosure provides for the modification of proteinsexpressed in a cell by the addition of one or more amino acid sequencemotifs that enhance transport of the proteins to the plasma membranes ofmammalian cells. Light-activated proteins having components derived fromevolutionarily simpler organisms may not be expressed or tolerated bymammalian cells, or may exhibit impaired subcellular localization whenexpressed at high levels in mammalian cells. Consequently, in someembodiments, the subject light-activated anion channel proteins that areexpressed in a cell can be fused to one or more amino acid sequencemotifs selected from the group consisting of a signal peptide, anendoplasmic reticulum (ER) export signal, a membrane trafficking signal,and/or an N-terminal golgi export signal. The one or more amino acidsequence motifs that enhance protein transport to the plasma membranesof mammalian cells can be fused to the N-terminus, the C-terminus, or toboth the N- and C-terminal ends of a protein in order to facilitateoptimal expression and/or localization of the protein in the plasmamembrane of a cell. Optionally, the subject light-activated anionchannel proteins and the one or more amino acid sequence motifs may beseparated by a linker. In some embodiments, the light-activated anionchannel protein can be modified by the addition of a trafficking signal(ts) which enhances transport of the protein to the cell plasmamembrane. In some embodiments, the trafficking signal can be derivedfrom the amino acid sequence of the human inward rectifier potassiumchannel Kir2.1. In other embodiments, the trafficking signal cancomprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:83).

Trafficking sequences that are suitable for use can comprise an aminoacid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:83)).

A trafficking sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Signal sequences that are suitable for use can comprise an amino acidsequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, amino acid sequence identity to an amino acid sequence such as oneof the following:

1) the signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:85))

2) the β2 subunit signal peptide of the neuronal nicotinic acetylcholinereceptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:86));

3) a nicotinic acetylcholine receptor signal sequence (e.g.,MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:87)); and

4) a nicotinic acetylcholine receptor signal sequence (e.g.,MRGTPLLLVVSLFSLLQD (SEQ ID NO:88)).

A signal sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Endoplasmic reticulum (ER) export sequences that are suitable for usewith a light-activated anion channel protein of the present disclosureinclude, e.g., VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:89); VLGSL (SEQ ID NO:90); etc.); NANSFCYENEVALTSK (SEQ ID NO:91);FXYENE (SEQ ID NO:92) (where X is any amino acid), e.g., FCYENEV (SEQ IDNO:93); and the like. An ER export sequence can have a length of fromabout 5 amino acids to about 25 amino acids, e.g., from about 5 aminoacids to about 10 amino acids, from about 10 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, or fromabout 20 amino acids to about 25 amino acids.

In some embodiments, the signal peptide sequence in the protein can bedeleted or substituted with a signal peptide sequence from a differentprotein.

Polynucleotides and Vectors

Aspects of the present disclosure include nucleic acids, such aspolynucleotides, that comprise a nucleotide sequence that encodes one ormore of the subject proteins described herein (e.g., one or morelight-activated anion channel proteins as described above). In someembodiments, a subject polynucleotide comprises an expression cassette,wherein the expression cassette contains a plurality of components(e.g., a plurality of coding sequences) that are utilized to express oneor more proteins encoded by the polynucleotide in a target cell.

In some embodiments, a portion of a polynucleotide encoding a subjectprotein is operably linked to a promoter sequence. Any suitable promoterthat functions in a target cell can be used for expression of thesubject polynucleotides. In certain embodiments, a promoter sequence canbe a promoter that is specific to a particular target cell type or to aparticular tissue type, such as a particular neuron or a pan-neuronalpromoter. Initiation control regions of promoters, which are useful todrive expression of polynucleotides in a specific animal cell, arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving expression of the subject polynucleotidescan be used. In some embodiments, the promoter used to drive expressionof a subject protein can be the Thyl promoter (See, e.g., Llewellyn, etal., 2010, Nat. Med., 16(10):1161-1166). In some embodiments, thepromoter used to drive expression of a subject protein can be a humansynapsin (hSyn) promoter, a human elongation factor 1-α (EF1α) promoter,a cytomegalovirus (CMV) promoter, a CMV early enhancer/chicken β actin(CAG) promoter, a synapsin-I promoter (e.g., a human synapsin-Ipromoter), a human synuclein 1 promoter, a human Thyl promoter, acalcium/calmodulin-dependent kinase II alpha (CAMKIIa) promoter, or anyother promoter capable of driving expression of the a subject nucleicacid sequence in a target cell.

In some embodiments, a promoter may be an inducible promoter. Forexample, the promoter may be induced by a trans-acting factor thatresponds to an exogenously administered drug. Examples of induciblepromoters include, but are not limited to, tetracycline-on ortetracycline-off promoters, or tamoxifen-inducible CreER.

In some embodiments, a subject polynucleotide may comprise a ribosomalskip sequence that can be used to generate two separate proteins fromthe same transcript. In such embodiments, a subject polynucleotide willtypically include a coding sequence that encodes a light-activatedprotein as well as a response protein. In these embodiments, a ribosomalskip sequence may be placed between the two coding sequences to producetwo distinct proteins (namely, the light-activated protein and theresponse protein) from the same transcript.

Also provided herein are recombinant expression vectors comprising thesubject polynucleotides or any variant thereof as described herein.Vectors according to the present disclosure also include vectorscomprising a nucleotide sequence that encodes an RNA (e.g., an mRNA)that when transcribed from the polynucleotides of the vector will resultin the accumulation of a subject protein on the plasma membranes oftarget cells. Vectors which may be used include, without limitation,lentiviral, HSV, adenoviral, and adeno-associated viral (AAV) vectors.Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV andEIAV. Lentiviruses may be pseudotyped with the envelope proteins ofother viruses, including, but not limited to VSV, rabies, Mo-MLV,baculovirus and Ebola. Such vectors may be prepared using standardmethods in the art.

In some embodiments, a vector may be a recombinant AAV vector. AAVvectors are DNA viruses of relatively small size that can integrate, ina stable and site-specific manner, into the genome of the cells thatthey infect. They are able to infect a wide spectrum of cells withoutinducing any effects on cellular growth, morphology or differentiation,and they do not appear to be involved in human pathologies. The AAVgenome has been cloned, sequenced and characterized. It encompassesapproximately 4700 bases and contains an inverted terminal repeat (ITR)region of approximately 145 bases at each end, which serves as an originof replication for the virus. The remainder of the genome is dividedinto two essential regions that carry the encapsidation functions: theleft-hand part of the genome that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (see, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 5-14,Hudder Arnold, London, U K (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006),the disclosures of each of which are hereby incorporated by referenceherein in their entireties). Methods for purifying for vectors may befound in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and6,995,006 and WO/1999/011764 titled “Methods for Generating High TiterHelper-free Preparation of Recombinant AAV Vectors”, the disclosures ofwhich are herein incorporated by reference in their entirety. Methods ofpreparing AAV vectors in a baculovirus system are described in, e.g., WO2008/024998. AAV vectors can be self-complementary or single-stranded.Preparation of hybrid vectors is described in, for example, PCTApplication No. PCT/US2005/027091, the disclosure of which is hereinincorporated by reference in its entirety. The use of vectors derivedfrom the AAVs for transferring genes in vitro and in vivo has beendescribed (See e.g., International Patent Application Publication Nos.:91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and5,139,941; and European Patent No.: 0488528, all of which are herebyincorporated by reference herein in their entireties). Thesepublications describe various AAV-derived constructs in which the repand/or cap genes are deleted and replaced by a gene of interest, and theuse of these constructs for transferring the gene of interest in vitro(into cultured cells) or in vivo (directly into an organism). Thereplication-defective recombinant AAVs according to the presentdisclosure can be prepared by co-transfecting a plasmid containing thenucleic acid sequence of interest flanked by two AAV inverted terminalrepeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes(rep and cap genes), into a cell line that is infected with a humanhelper virus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

In some embodiments, the vector(s) for use in the methods of the presentdisclosure are encapsidated into a virus particle (e.g. AAV virusparticle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, andAAV16). Accordingly, the present disclosure includes a recombinant virusparticle (recombinant because it contains a recombinant polynucleotide)comprising any of the vectors described herein. Methods of producingsuch particles are known in the art and are described in U.S. Pat. No.6,596,535, the disclosure of which is hereby incorporated by referencein its entirety.

In some embodiments, the subject anion channel proteins can be combinedwith various promoters and/or fluorescent proteins (XFP) for targetingspecific neuronal populations in mammalian brains. For example, thefollowing adeno associated vectors (AAVs) and components thereof may beused without limitation: AAV-CamKII-iChR-XFP, AAV-hSyn-iChR-XFP,AAV-mThy1-iChR-XFP, AAVmThy1-iChR-XFP, AAV-GFAP-iChR-XFP,AAV-VGAT-iChR-XFP, AAV-PET1-iChR-XFP, AAV-NPY-iChR-XFP,AAV-SST-iChR-XFP, AAV-AVP5.5-iChR-XFP, AAV-Ef1a-iChR-XFP,AAV-FLEX-rev-iChR-XFP, AAV-CAG-iChR-XFP, AAV-CAG-FLEX-iChR-XFP. OtherAAV vectors that may be used in association with the polynucleotidesinclude those with double floxed inverted reading frames (DIO) whichallow expression of proteins under the control of recombinases such asas Cre and Flp: AAV-Ef1a-DIO(Cre)-iChR-XFP (Cre-dependent expression),AAV-Ef1a-DIO(Flp)-iChR-XFP (Flp-dependent expression),AAV-Ef1a-DIO(Cre)-DIO(Flp)-iChR-XFP (Cre and Flp dependent expression).

Another major viral transduction system utilizes lentivirus includingthe following potential expression vectors: pLenti-CamKII-iChR-XFP,pLenti-Ef1a-iChR-XFP, pLenti-mThy1-iChR-XFP, pLenti-hThy 1-iChR-XFP,pLenti-hSyn-iChR-XFP, pLenti-VGAT-iChR-XFP, pLenti-Hcrt-iChRXFP. Herpessimplex virus (HSV) can be utilized to transport proteins of interestover synapses (anterograde) which includes the following expressionvectors: HSV-EF1a-iChR-XFP and HSVEF1a-DIO-iChR-XFP. Rabies andpseudorabies virus can be utilized for retrograde transports oversynapses using the following expression vector: SAD(delta)G-iChR-XFP andSAD(delta)G-DIOiChR-XFP. Other mammalian expression vectors include:pcDNA3.1-CMV-iChR-XFP and pCAGGS-iChR-XFP.

Neuron-specific promoters and other control elements (e.g., enhancers)are known in the art. Suitable neuron-specific control sequencesinclude, but are not limited to, a neuron-specific enolase (NSE)promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. No.6,649,811, U.S. Pat. No. 5,387,742); an aromatic amino aciddecarboxylase (AADC) promoter; a neurofilament promoter (see, e.g.,GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBankHUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1161); a serotoninreceptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylasepromoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) andNeuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al.,Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see,e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter(see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652(1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J.17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMVenhancer/platelet-derived growth factor-β promoter (see, e.g., Liu etal. (2004) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2004)Development 131:3295-3306); and an alpha subunit ofCa(²⁺)-calmodulin-dependent protein kinase II (CaMKIIα) promoter (see,e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250).

Host Cells

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce an opsin of the present disclosure.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable mammalian cells include primary cells and immortalized celllines. In some cases, the mammalian cell is a neuron, e.g., anon-immortalized (primary) neuron. In other cases, the mammalian cell isan immortalized cell line.

Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. Suitable cell lines include, but are not limited to, ahuman glioma cell line, e.g., SVGp12 (ATCC CRL-8621), CCF-STTG1 (ATCCCRL-1718), SW 1088 (ATCC HTB-12), SW 1783 (ATCC HTB-13), LLN-18 (ATCCCRL-2610), LNZTA3WT4 (ATCC CRL-11543), LNZTA3WT11 (ATCC CRL-11544),U-138 MG (ATCC HTB-16), U-87 MG (ATCC HTB-14), H4 (ATCC HTB-148), andLN-229 (ATCC CRL-2611); a human medulloblastoma-derived cell line, e.g.,D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185); ahuman tumor-derived neuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060),SK-N-DZ (ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCCCRL-2142), IMR-32 (ATCC CCL-127), etc.; a mouse neuronal cell line,e.g., BC3H1 (ATCC CRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCCCRL-2534), C8-S(ATCC CRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCCCCL-147), SW10 (ATCC CRL-2766), NG108-15 (ATCC HB-12317); a rat neuronalcell line, e.g., PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC CRL-2006), C6(ATCC CCL-107), F98 (ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCCCRL-2754), R3 (ATCC CRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the present invention include, but are not limitedto, Salmonella typhi and S. typhimurium. Suitable Shigella strainsinclude, but are not limited to, Shigella flexneri, Shigella sonnei, andShigella disenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides,Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and thelike. In some embodiments, the host cell is Escherichia coli.

Pharmaceutical Compositions

Aspects of the disclosure include pharmaceutical compositions thatcomprise the subject polynucleotides, vectors, or components thereof.The subject pharmaceutical compositions may be administered to a subjectfor purposes of genetically modifying a target cell so that the targetcell expresses one or more of the subject proteins. A subjectpharmaceutical composition may, in some embodiments, comprise apharmaceutically acceptable excipient. In some embodiments, apharmaceutical composition may comprise components to facilitatedelivery of the subject polynucleotides or vectors to a target cell,including but not limited to transfection reagents or componentsthereof, such as lipids, polymers, and the like.

In some embodiments, a subject pharmaceutical composition will besuitable for injection into a subject, e.g., will be sterile. Forexample, in some embodiments, a subject pharmaceutical composition willbe suitable for injection into a subject, e.g., where the composition issterile and is free of detectable pyrogens and/or other toxins.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public as well, andmay be incorporated into the pharmaceutical compositions of the presentdisclosure without limitation.

Target Cells and Tissues

As summarized above, aspects of the present disclosure includedelivering the subject polynucleotides, or components thereof, to targetcells. Target cells are generally cells that carry or transmitelectrical impulses, such as nerve cells. In some embodiments, a targetcell may be, e.g., a sensory neuron, a motor neuron, or an interneuron.Target cells of the disclosure may include cells of the central nervoussystem and/or cells of the peripheral nervous system. In someembodiments, a target tissue may include a plurality of nerve fibers, anerve, a nerve cell ganglion, a neuromuscular junction, a tissue that isinnervated by nerves, including but not limited to muscle, skin, orendocrine tissue, or an anatomical region, such as a portion orsub-portion of the brain or spinal cord. In some embodiments, a targettissue may be a portion of an individual cell, such as specific axon ofa nerve cell.

Once the subject polynucleotides have been delivered to a target cell ortissue, the polynucleotides enter the target cells and are expressed. Insome embodiments, the subject polynucleotides may containtissue-specific promoters so that expression only occurs in target cellswherein the tissue-specific promoter is active. In this way, if asubject polynucleotide is delivered to cells other than a target cell,the polynucleotide will not be expressed in the non-target cells becausethe tissue-specific promoter will be inactive in those cells. In someembodiments, a subject polynucleotide may contain an inducible promoter,such that expression of the polynucleotide only takes place when anexogenously administered drug is present is a sufficient concentrationwithin the cell to activate the promoter.

Systems and Devices

Aspects of the present disclosure include systems and devices that canbe used to carry out aspects of the subject methods. The subject systemsgenerally include an engineered light-activated anion channel protein,as described above, and one or more devices for delivering light of anactivating wavelength to a target tissue or cell. Devices that find usein the subject methods include delivery devices that can be used todeliver the subject polynucleotides to target cells and tissues,light-generating devices that can be used to illuminate target cellsthat express the subject light-activated proteins, and control devicesthat can be used to control the delivery of light to specific targetcells or tissues. Each of these components is further described below.

Delivery Devices

Aspects of the present disclosure include delivery devices that can beused to deliver a subject pharmaceutical composition to a target cell.The subject delivery devices may provide regular, irregular, programmed,or clinician- or patient-activated doses of the subject pharmaceuticalcompositions to one or more target cells to ensure that the target cellscontinue to express the subject protein(s) for a desired period of time.

The subject delivery devices may generally include various components,such as reservoirs, pumps, actuators, tubing components, needles,catheters, and any other suitable components for delivering the subjectpharmaceutical compositions to a target cell or tissue of a patient.Delivery devices may also include components that facilitatecomputerized operation, such as a power source, a processor comprising amemory, a user input device, and/or a graphical user interface. In someembodiments, a delivery device may be completely or partiallyimplantable within a patient. In some embodiments, a delivery device maybe operated by a caregiver, wherein the device is introduced into aportion of the patient's body, e.g., into the patient's brain, and asubject pharmaceutical composition is delivered to a target tissue,e.g., a portion of the patient's brain. In some embodiments, followingdelivery of the pharmaceutical composition, the device may be removed.In other embodiments, the device may be kept in place for later deliveryof additional pharmaceutical compositions.

Light-Generating Devices

Aspects of the present disclosure include light-generating devices thatcan be used to deliver light to target cells that express one or more ofthe subject proteins. Light-generating devices in accordance withembodiments of the present disclosure can generally produce light of avariety of different wavelengths from one or more light sources on thedevice. In some embodiments, a light-generating device may include alight cuff or sleeve that can be placed around or near target cellsexpressing one or more of the subject proteins. In some embodiments, aportion of the light source or the entire light source may beimplantable. The subject light-generating devices may be of any usefulconfiguration for stimulating the light-activated proteins disclosedherein. In some embodiments, for example, a light-generating device maycomprise components that facilitate exclusive illumination of a targetcell or tissue. For example, in some embodiments, a light-generatingdevice may exclusively direct light to a target cell, a portion of atarget cell, e.g., a particular axon of a nerve cell, or a specificanatomical structure, such as, e.g. a bundle of nerve fibers, a targettissue, or a portion of the spinal cord. By “exclusively direct light”is meant that the light-generating device only delivers light to thespecific target structure, and does not illuminate other structures. Forexample, in some embodiments, a light-generating device may beconfigured to illuminate an axon of a nerve cell, but not to illuminateany other portion of the nerve cell. In this way, the light from thelight-generating device only affects light-activated proteins in thespecific target structure that is illuminated.

Aspects of the disclosure include light delivery devices that includeone or more optical sources that are configured to deliver light in oneor more 2-dimensional and/or 3-dimensional patterns to one or moretarget locations, including but not limited to one or more portions(e.g., multiple layers) of a target tissue and/or anatomical structure.In certain embodiments, a light delivery device may include a pluralityof light sources (e.g., a plurality of laser light sources, LEDs, andthe like), as well as any suitable number of light guides that areconfigured to bend or shape light in a desired manner. Examples of lightdelivery devices are provided in U.S. Pat. No. 8,545,543, the disclosureof which is hereby incorporated by reference in its entirety.

In some embodiments, a light-generating device may not completelysurround the region containing a target cell expressing alight-activated protein, but, rather, can have a U-shape. In someembodiments, a light-generating device can have an attachment arm thatcan be used to guide the light-generating device to a specific region ortarget structure, e.g., a specific neuronal region. The attachment armcan be removed following implantation of the light-generating device orcan be left in place to fix the position of the light-generating devicein proximity to the target cells of interest.

In some embodiments, the subject light-generating devices may comprisean inner body, the inner body having at least one means for generatinglight which is connected to a power source. In some embodiments, thepower source can be an internal battery for powering thelight-generating device. In some embodiments, an implantablelight-generating device may comprise an external antenna for receivingwirelessly transmitted electromagnetic energy from an external sourcefor powering the device. The wirelessly transmitted electromagneticenergy can be a radio wave, a microwave, or any other electromagneticenergy source that can be transmitted from an external source to powerthe light-generating device. In some embodiments, the light-generatingdevice is controlled by, e.g., an integrated circuit produced usingsemiconductor or other processes known in the art.

In some embodiments, the light-generating device may comprise a lightemitting diode (LED). In some embodiments, the LED can generate blueand/or green light. In other embodiments, the LED can generate amberand/or yellow light. In some embodiments, several micro LEDs areembedded into the inner body of the light-generating device. In otherembodiments, the light-generating device is a solid state laser diode orany other means capable of generating light. The light-generating devicecan generate light having a wavelength and intensity sufficient toactivate a subject light-activated protein. In some embodiments, alight-generating device produces light having an intensity of any ofabout 0.05 mW/mm², 0.1 mW/mm², 0.2 mW/mm², 0.3 mW/mm², 0.4 mW/mm², 0.5mW/mm², about 0.6 mW/mm², about 0.7 mW/mm², about 0.8 mW/mm², about 0.9mW/mm², about 1.0 mW/mm², about 1.1 mW/mm², about 1.2 mW/mm², about 1.3mW/mm², about 1.4 mW/mm², about 1.5 mW/mm², about 1.6 mW/mm², about 1.7mW/mm², about 1.8 mW/mm², about 1.9 mW/mm², about 2.0 mW/mm², about 2.1mW/mm², about 2.2 mW/mm², about 2.3 mW/mm², about 2.4 mW/mm², about 2.5mW/mm², about 3 mW/mm², about 3.5 mW/mm², about 4 mW/mm², about 4.5mW/mm², about 5 mW/mm², about 5.5 mW/mm², about 6 mW/mm², about 7mW/mm², about 8 mW/mm², about 9 mW/mm², or about 10 mW/mm², inclusive,including values in between these numbers. In some embodiments, thelight-generating device produces light having an intensity of at leastabout 10 Hz, such as up to about 25 Hz, such as up to about 50 Hz, suchas up to about 75 Hz, such as up to about 100 Hz.

The subject light-generating devices are generally capable of generatinglight having a wavelength ranging from about 350 nm, up to about 360 nm,up to about 370 nm, up to about 380 nm, up to about 390 nm, up to about400 nm, up to about 410 nm, up to about 420 nm, up to about 430 nm, upto about 440 nm, up to about 450 nm, up to about 460 nm, up to about 470nm, up to about 475 nm, up to about 480 nm, up to about 490 nm, up toabout 500 nm, up to about 510 nm, up to about 520 nm, up to about 530nm, up to about 540 nm, up to about 550 nm, up to about 560 nm, up toabout 570 nm, up to about 580 nm, up to about 590 nm, up to about 600nm, up to about 610 nm, up to about 620 nm, up to about 630 nm, up toabout 635 nm, up to about 640 nm, up to about 650 nm, up to about 660nm, up to about 670 nm, up to about 680 nm, up to about 690 nm, up toabout 700 nm, up to about 710 nm, up to about 720 nm, up to about 730nm, up to about 740 nm, and/or up to about 750 nm.

In some embodiments, a light generating device may generate violet lighthaving a wavelength ranging from about 400 nm to about 475 nm. In someembodiments, a light generating device may generate blue light having awavelength ranging from about 475 nm to about 500 nm. In someembodiments, a light generating device may generate green light having awavelength ranging from about 500 nm to about 560 nm. In someembodiments, a light generating device may generate yellow light havinga wavelength ranging from about 560 nm to about 590 nm. In someembodiments, a light generating device may generate orange light havinga wavelength ranging from about 590 nm to about 620 nm. In someembodiments, a light generating device may generate red light having awavelength ranging from about 620 nm to about 650 nm.

In some embodiments, a subject light-generating device may include oneor more optical fibers that can transmit light from a light source anddeliver the light to a target structure. The optical fibers may compriseplastic or glass materials, and in some embodiments may be suitablyflexible to facilitate placement of the light-generating device inlocations that could not be accommodated by rigid structures. Forexample, in some embodiments, a light-generating device may comprise alight source that generates light, as well as one or more optical fibersthat can be placed in various locations on or in the patient's body.Light from the light source can pass through the optical fiber, passingaround corners and bends in the optical fiber, and emerge at the end ofthe optical fiber to deliver light to a target structure.

In some embodiments, the subject light-generating devices may comprise aplurality of light sources that can be used to illuminate a targettissue with different wavelengths of light. For example, in someembodiments, a light-generating device may comprise a first light sourcethat generates light of a first wavelength, e.g., red light, and asecond light source that generates light of a second wavelength, e.g.,blue light. Such light-generating devices may be used to simultaneouslyilluminate the same target tissue with light of both wavelengths, or mayalternately illuminate the target tissue with light of the firstwavelength and light of the second wavelength. In some embodiments, suchlight generating devices may be used to deliver light from the samelight source to different target tissues. For example, in someembodiments a light-generating device may deliver light of a firstwavelength to a first target tissue, and may deliver light of a secondwavelength to a different target tissue.

Control Devices

Aspects of the disclosure include a controller, processor (e.g., acomputer) and computer readable medium that are configured or adapted tocontrol or operate one or more components of the subject systems. Insome embodiments, a system includes a controller that is incommunication with one or more components of the systems, as describedherein, and is configured to control aspects of the systems and/orexecute one or more operations or functions of the subject systems. Insome embodiments, a system includes a processor and a computer-readablemedium, which may include memory media and/or storage media.Applications and/or operating systems embodied as computer-readableinstructions on computer-readable memory can be executed by theprocessor to provide some or all of the functionalities describedherein.

In some embodiments, a system includes a user interface, such as agraphical user interface (GUI), that is adapted or configured to receiveinput from a user, and to execute one or more of the methods asdescribed herein. In some embodiments, a GUI is configured to displaydata or information to a user.

Aspects of the present disclosure include control devices that cancontrol, or modulate, the amount of light that is emitted from thesubject light-generating devices. In some embodiments, a control devicemay be configured to modulate the wavelength and/or the intensity oflight that is delivered to a target tissue from a light-generatingdevice. In some embodiments, a control device may be configured tomodulate the frequency and/or duration of light that is delivered to atarget tissue from a light-generating device. For example, in someembodiments, a control device may be configured to deliver pulses oflight from the light-generating device to a target tissue. The controldevice can modulate the frequency and/or duration of the light pulsessuch that the target tissue is illuminated with light from thelight-generating device, e.g., at a regular or irregular rate, accordingto a user input, etc. In some embodiments, a control device can producepulses of light from the light-generating device that have a durationranging from about 1 millisecond or less, up to about 1 second, up toabout 10 seconds, up to about 20 seconds, up to about 30 seconds, up toabout 40 seconds, up to about 50 seconds, up to about 60 seconds ormore. In some embodiments, a control device can produce pulses of lightfrom the light-generating device that have a frequency of 1 pulse permillisecond, up to about 1 pulse per second, up to about 1 pulse perminute, up to about 1 pulse per 10 minutes, up to about 1 pulse per 20minutes, up to about 1 pulse per 30 minutes.

In some embodiments, a subject control device may comprise a powersource that can be mounted to a transmitting coil. In some embodiments,a battery can be connected to the power source for providing powerthereto. A switch can be connected to the power source, allowing anoperator (e.g., a patient or caregiver) to manually activate ordeactivate the power source. In some embodiments, upon activation of theswitch, the power source can provide power to the light-generatingdevice through electromagnetic coupling between the transmitting coil onthe control device and an external antenna of an implantablelight-generating device (such as a light cuff or sleeve). Thetransmitting coil can establish an electromagnetic coupling with theexternal antenna of the implantable light-generating device when inproximity thereof, for supplying power to the light-generating deviceand for transmitting one or more control signals to the light-generatingdevice. In some embodiments, the electromagnetic coupling between thetransmitting coil of the control device and the external antenna of theimplantable light-generating device can be radio-frequency magneticinductance coupling. When radio-frequency magnetic inductance couplingis used, the operational frequency of the radio wave can be betweenabout 1 and 20 MHz, inclusive, including any values in between thesenumbers (for example, about 1 MHz, about 2 MHz, about 3 MHz, about 4MHz, about 5 MHz, about 6 MHz, about 7 MHz, about 8 MHz, about 9 MHz,about 10 MHz, about 11 MHz, about 12 MHz, about 13 MHz, about 14 MHz,about 15 MHz, about 16 MHz, about 17 MHz, about 18 MHz, about 19 MHz, orabout 20 MHz). However, other coupling techniques may be used, such asan optical receiver, infrared, or a biomedical telemetry system (See,e.g., Kiourti, “Biomedical Telemetry: Communication between ImplantedDevices and the External World, Opticon 1826, (8): Spring, 2010).

Turning now to FIG. 8, a first example of an optical stimulation system100 is depicted. The optical stimulation system 100 comprises a deliverydevice 101 for delivering a subject polynucleotide to a target tissue,e.g., brain tissue 107 of a patient. Also provided are alight-generating device 102, a control device 103, and optical fibers104 for conveying light generated by the light-generating device 102 toa light array 105 positioned on a light cuff 106.

Turning now to FIG. 9, a second example of an optical stimulation system110 is depicted. The optical stimulation system 110 comprises a catheter112 for delivering a subject polynucleotide to a target tissue, e.g.,brain tissue 107 of a patient. Also provided are a light-generatingdevice 102, a control device 103, and optical fibers 104 for conveyinglight generated by the light-generating device 102 to the end of theoptical fibers 104.

Turning now to FIG. 10, a third example of an optical stimulation system120 is depicted. The optical stimulation system 120 comprises alight-generating device 102, a control device 103, and optical fibers104 for conveying light generated by the light generating device 102 tovarious positions along the spinal cord 121 of the patient.

Methods

Aspects of the present disclosure include methods for optogeneticmodulation of action potentials in target cells. The subject methodsgenerally involve introducing a light-activated anion channel proteininto a target cell and illuminating the target cell with light of anactivating wavelength. Illumination of the target cell with light of anactivating wavelength causes the light-activated anion channel proteinto allow one or more anions to pass through the plasma membrane of thetarget cell. The passage of the anions through the plasma membrane ofthe target cell has a desired effect, such as, e.g., modulating themembrane potential of the plasma membrane. In some embodiments, thepassage of the anion species through the plasma membrane may be used tomodulate one or more neurological responses or processes in a patient,and may therefore by used to treat a disease or condition in thepatient. As such, in some embodiments, the subject methods involvetreating a patient for a condition, such as a neurological condition,using the systems and devices provided herein. The subject methods arenow described in greater detail below.

Modulating Membrane Potentials in Target Cells

In some embodiments, the subject methods involve modulating membranepotentials in target cells using the subject systems and devices. Insome embodiments, a nucleic acid encoding a subject light-activatedanion channel protein is introduced into a target cell such that thetarget cell expresses the protein. The target cell is then illuminatedwith light of an activating wavelength using a light-generating device.Illumination of the light-activated anion channel protein results in themovement of one or more anions through the plasma membrane of the cellin response to light. In some embodiments, for example, thelight-activated anion channel protein is a chloride anion channelprotein, and in response to light the anion channel protein allowschloride ions to flow from the external side of the plasma membrane tothe internal side of the plasma membrane. In certain embodiments, theactivation of a light-activated chloride anion channel results inhyperpolarization of a cell. In certain embodiments, the activation of alight-activated chloride anion channel results in hyperpolarization of anerve cell membrane and inhibition of action potentials withoutdepolarizing the nerve cell to or beyond an action potential generationthreshold.

Specific Inhibition of Activity Along an Axonal Projection

In some embodiments, the subject methods involve inhibiting and/orblocking activity along a portion of a nerve cell (e.g., along an axonof a nerve cell, or at the termination of an axonal projection of anerve cell) using the subject systems and devices. For example, in someembodiments, the subject methods involve introducing into a nerve cell asubject light-activated anion channel protein. Polynucleotides encodingthe proteins are introduced into the nerve cell, and the proteins areexpressed by the nerve cell and inserted into the plasma membrane of thenerve cell.

Next, a light-generating device is positioned such that a target portionof the nerve cell (e.g., the axon, or a portion of the axon of the nervecell) is illuminated with light of an activating wavelength when thelight-generating device is activated. Next, the light-generating deviceis activated to deliver light to the desired nerve cell or portionthereof to cause the light-activated anion channel protein to allowanions to flow through the plasma membrane of the nerve cell. In someembodiments, the light-activated anion channel protein is a chlorideanion channel protein that allows chloride anions to flow from theexternal side of the nerve cell membrane to the internal side of thenerve cell membrane. This results in hyperpolarization of the plasmamembrane of the nerve cell without depolarizing the nerve cell membraneto or beyond an action potential generation threshold.

Hyperpolarization of the plasma membrane of the nerve cell inhibitsaction potentials by increasing the stimulus that is required todepolarize the membrane to an action potential threshold. Accordingly,the subject methods may be used to block or inhibit action potentials ina particular nerve cell or in a portion thereof (e.g., an axon or aportion thereof) by delivering light of an activating wavelength to thenerve cell or to a specific portion of the nerve cell. Importantly,action potentials may still propagate through other portions of thenerve cell or axon that are not illuminated with light of a wavelengththat activates the subject light-activated anion channel protein. Inthis way, specificity is achieved for inhibiting action potentials inspecific target cells or specific portions thereof.

Modeling of Diseases or Conditions Involving Action Potentials

In some embodiments, the subject methods may be used for studying and/ormodeling certain diseases or conditions in a subject, such as conditionsthat involve or result from improper formation of action potentialsand/or an improper blockade of action potential formation within a cell.For example, the subject methods may be used to specifically inhibit theformation of action potentials in target cells, such as specific targetnerve cells, to study the effects of blocking action potential formationin those cells. In some embodiments, the subject methods may be used toselectively inhibit the formation of action potentials in certainportions of a target cell, such as an axon of a target nerve cell, tostudy the effects of inhibiting action potential formation in theselected portion of the target cell. Such methods may be used as modelsof diseases or conditions in which action potentials fail to properlyform in a target cell or a portion thereof, or wherein action potentialsare erroneously formed in a target cell or a portion thereof.

In some embodiments, the subject methods may be used, e.g., forscreening compounds that may be effective in treating diseases orconditions involving the formation of action potentials in target cells,or the failure of action potentials to form in target cells. In someembodiments, the screening methods involve culturing cells in vitro andcontacting the cultured cells with a nucleic acid encoding one or moreof the subject engineered light-activated anion channel proteins suchthat the cultured cells express the anion channel protein. A culturedcell expressing the anion channel protein is contacted with a testcompound, and the cell is then exposed to light of an activatingwavelength to inhibit the formation of action potentials within the cellor a portion thereof. The ability of the test compound to elicit adesired effect or response from the cell while action potentialformation is being inhibited may be useful in the treatment of aparticular disease or condition.

In some embodiments, the subject methods may be used in animal models(including but not limited to transgenic animal models) of diseases ofconditions associated with improper formation of action potentialswithin target cells, or portions thereof, or associated with theblockade of the formation of action potentials within target cells, orportions thereof. For example, in some embodiments, a target cell of ananimal (such as a nerve cell, e.g., a brain cell of a rodent) may becontacted with a nucleic acid encoding a subject engineeredlight-activated anion channel protein so that the anion channel proteinis expressed by the target cell. Next, the target cell is illuminatedwith light of an activating wavelength to inhibit the formation ofaction potentials in the target cell. The effect of the inhibition ofaction potential formation within the target cell, or a portion thereof,on the animal can then be examined. The use of transgenic animals thatoverexpress one or more gene products, or the use of “knock-out”transgenic animals that fail to express one or more gene products, maybe used to investigate the role of specific gene products in theformation of action potentials in target cells.

Methods of Treatment

In some cases, a subject method involves modulating the activity of atarget cell in vivo. A nucleic acid comprising a nucleotide sequenceencoding a subject light-responsive anion channel polypeptide isintroduced into a target cell, where the encoded light-responsive anionchannel polypeptide is produced in the cell; and the light-responsiveanion channel polypeptide is activated by exposure to light of anactivating wavelength.

In some embodiments, the subject methods are used to treat a patient fora condition or disorder, such as a neurological condition or disorder,by optogenetically modulating the action potentials of target cellswithin the patient. In some embodiments, the subject methods involveintroducing an engineered light-activated anion channel protein into atarget tissue within the patient. In some embodiments, introduction ofthe subject anion channel proteins into the target tissue isaccomplished using a subject delivery device. The polynucleotidesencoding the subject anion channel proteins are introduced into thetarget tissue, and the proteins are expressed by target cells (e.g.,nerve cells) in the target tissue and inserted into the plasma membraneof the target cells.

Next, a light-generating device is positioned to illuminate the targettissue with light of an activating wavelength when the light-generatingdevice is activated. The light-generating device is activated (either bythe patient, or by a caregiver (e.g., medical personnel)) to deliverlight to the target tissue to cause the light-activated anion channelproteins to allow anions (e.g., chloride anions) to pass through theplasma membrane and hyperpolarize the plasma membrane, thus inhibitingthe formation of action potentials within the cell(s) of the targettissue.

As such, the formation of action potentials within the cell is blockedfor the duration of the effect of the light pulse and the resultinghyperpolarization of the plasma membrane. Accordingly, the subjectmethods may be used to block the formation of an action potential in anerve cell by introducing the subject anion channel proteins into thenerve cell and illuminating the nerve cell with light of an activatingwavelength from a light-generating device. As the duration of the actionpotential blockade can be tailored to outlast the duration of a lightpulse, inhibition of action potential formation may be achieved usingpulsed light delivery, rather than continuous light delivery.

In some embodiments, the subject methods involve treating a subject fora disorder by inhibiting the formation of action potentials in a targettissue. Accordingly, in some embodiments, the subject methods involvetreating a subject by introducing into a target cell a light-activatedanion channel protein. Polynucleotides encoding these proteins areintroduced into the target cell, and the proteins are expressed by thetarget cell and inserted into the plasma membrane of the target cell.Next, the target cell is illuminated with light of an activatingwavelength from a light-generating device to cause the light-activatedanion channel protein to allow anions (e.g., chloride anions) to flowthrough the plasma membrane from outside of the cell to the inside ofthe cell.

Once inside the cell, the chloride anions hyperpolarize the membrane toinhibit the formation of an action potential. The hyperpolarization ofthe membrane prevents the formation of an action potential and thereforeprevents the cell from, e.g., generating action potentials insurrounding cells, e.g., neighboring nerve cells; mediating the releaseof neurotransmitters, modulators, or hormones; mediating musclecontraction; and the like until the effect of the membranehyperpolarization dissipates. Accordingly, the subject methods may beused to treat a subject for a disorder by blocking the formation ofaction potentials within a target cell. Since the duration of themembrane hyperpolarization can be tailored to outlast the duration ofthe light pulse, inhibition of action potential formation may beachieved using pulsed light delivery, rather than continuous lightdelivery.

Accordingly, the subject methods may be used to treat any disease orcondition in which blocking or inhibiting the formation of an actionpotential a target cell, or along a particular portion of a target cell,would have a therapeutic effect for the patient. Examples of therapeuticapplications of the subject methods include, without limitation, therapyfor cardiac rhythm disorders, such as pacing, cardioversion,defibrillation, resynchronization, or other cardiac-related conditions;gastrointestinal therapy, such as therapy to address obesity, motilitydisorders (e.g., gastroparesis), dyspepsia, or other therapies, therapyfor pelvic floor tissue (e.g., sacral or pudendal nerve tissue) tosupport pelvic floor therapy such as pain therapy, urinary or fecalincontinence therapy, sexual dysfunction, or other therapies; cranialnerve therapy, such as therapy to relieve occipital neuralgia,trigeminal neuralgia, facial pain, migraine headaches; therapy for thetreatment of pain, such as nociceptive pain or neuropathic pain; therapyfor neurological and/or psychiatric conditions; therapy for endocrineconditions; or the like. Specificity can be achieved as above byinhibiting action potential formation in specific subdomains or portionsof the axonal arborization or cell.

Kits

Also provided are kits that at least include the subject systems anddevices or components thereof, e.g., as described above, andinstructions for how to use the subject systems and/or devices tooptogenetically modulate action potentials in a target tissue. In someembodiments, a kit may include one or more of the subjectpolynucleotides, vectors, or pharmaceutical compositions. Kits inaccordance with embodiments of the present disclosure may also includeone or more devices, such as one or more delivery devices, one or morelight-generating devices, and/or one or more control devices.

The instructions for using the systems and devices as discussed aboveare generally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e. associated with the packaging or sub-packaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer-readable storage medium, e.g.,a digital storage medium, e.g., a CD-ROM, diskette, etc. Theinstructions may take any form, including complete instructions for howto use the systems and devices or as a website address with whichinstructions posted on the Internet may be accessed.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Materials and Methods

The following materials and methods were used in the examples below.

Structural Representations

Figures of the C1C2 structure were generated using the PyMOL MolecularGraphics System, Version 1.7.0.1 (Schrödinger, LLC; PNAS 98:10037-41,2001).

Point Mutagenesis of C1C2

C1C2 mutations were introduced using the QuickChange™ Site-Directedmutagenesis kit (Agilent) and purified with QIAprep™ Spin Miniprep Kits(Qiagen) after transformation and amplification in E. coli. AAV vectorsbearing the CaMKIIα promoter were used for protein expression inneurons, and a pcDNA3.1 vector bearing the CMV promoter was used forexpression in HEK cells. All clones were fused to the eYFP (enhancedyellow fluorescent protein) gene for fluorescence microscopy.

Neuronal Culture Preparation and Imaging

Primary hippocampal neurons were cultured from postnatal day 0 (P0)Sprague-Dawley rat pups (Charles River). The CA1 and CA3 regions wereisolated, digested with 0.4 mg/mL papain (Worthington), and plated onto12 mm glass coverslips pre-coated with 1:30 Matrigel (Beckton DickinsonLabware), at a density of 65,000 cells per well in 24-well plates. Thecultured cells were maintained in Neurobasal-A medium (Invitrogen)containing 1.25% FBS (HyClone), 4% B-27 supplement (Gibco), 2 mMGlutamax (Gibco) and 2 mg/ml fluorodeoxyuridine (FUDR, Sigma), and keptin a humid culture incubator with 5% CO₂ at 37° C.

Cells were transfected at 6-10 days in vitro (DIV). A DNA-CaCl₂ mixcontaining the following was prepared per each well to be transfected: 2μg of DNA (prepared using an endotoxin-free preparation kit (Qiagen))1.875 μl 2M CaCl₂, and sterile water to a total volume of 15 μl. Anadditional 15 μl of 2× filtered HEPES-buffered saline (HBS, in mM: 50HEPES, 1.5 Na₂HPO₄, 280 NaCl, pH 7.05 with NaOH) was added per DNA-CaCl₂mix, and the resulting DNA-CaCl₂-HBS mix was incubated at roomtemperature for 20 minutes. During this time, the neuronal growth mediumwas removed from the wells and saved at 37° C., and replaced with 400 μlpre-warmed minimal essential medium (MEM). Once the DNA-CaCl₂-HBS mixincubation was complete, the mix was then added dropwise into each well,and the plates were kept in the culture incubator for 45-60 minutes.Afterwards, each well was washed three times with 1 ml of pre-warmedMEM, and then the MEM was replaced with the original neuronal growthmedium. The transfected cells were then returned to the cultureincubator until recordings.

For confocal images of opsin-eYFP-expressing neurons, coverslips oftransfected cells expressing were fixed for 15 minutes in 4%paraformaldehyde and mounted with PVA-DABCO. Images were acquired with aLeica DM600B confocal microscope, and the same settings were used acrossimages.

Electrophysiological Recordings in Hippocampal Neurons

The Spectra X Light engine (Lumencor) was coupled to the fluorescenceport of an Olympus BX61WI microscope to detect eYFP expression and todeliver light for opsin activation. 475/15 and 632/22 filters were usedfor blue light and red light respectively, and light power densitythrough a 40× objective was measured with a power meter (ThorLabs).Whole-cell recordings were obtained with patch pipettes (4-6 MQ) pulledfrom glass capillaries (Sutter Instruments) with a horizontal puller(P-2000, Sutter Instruments). The external recording solution contained(in mM): 135 NaCl, 4 KCl, 10 HEPES, 2 CaCl₂, 2 MgCl₂, 30 D-glucose, pH7.3 with synaptic transmission blockers 2 μM D-APV, 1 μM NBQX. Theinternal solution recording solution contained (in mM): 140 K-gluconate,10 HEPES, 10 EGTA, 2 MgCl2, pH 7.3. Measurements were corrected for theliquid junction potential of +16 mV. We used 3 M CsCl agar bridges forthe reference electrode at all recordings. Series resistance wasmonitored throughout recordings for stability. Recordings were madeusing a MultiClamp700B amplifier (Molecular Devices). pClamp10.3(Molecular Devices), OriginLab8 (OriginLab), and Sigmaplot (SPSS)software was used to record and analyze data.

The stationary photocurrent upon light activation was used as themeasure of photocurrent amplitude at different membrane potentials. Thereversal potential (Vrev) was defined as the point where the stationaryphotocurrent amplitude was 0 pA. Action potential threshold was measuredat the voltage deflection point at which the first-order derivative ofthe membrane potential (dV/dt) exhibited a sharp transition,typically >10 mV/ms. The resting membrane potential of the cell wasmeasured in current-clamp after attaining whole-cell configuration.Input resistance was calculated from the steady-state current responsesevoked by 20 mV hyperpolarizing steps in voltage-clamp. To investigateaction potential inhibition, we tested opsin-expressing cells under twodifferent spike induction protocols. Spikes were electrically evokedwith intracellular current injections, either with short electricalpulses (30 ms pulse width, 50-280 pA) 10 Hz, or with a continuous 3 selectrical pulse. Light was applied for 1 s (during the 10 Hz train) or0.5 s (during the continuous pulse) during the middle of the electricalcurrent injection. Spike inhibition probability was calculated as thefraction of electrically-evoked spikes that were blocked during thelight pulse epoch of the electrical stimulation.

HEK Cell Culture Preparation

Human embryonic kidney cell cultures (HEK-293: ATCC® CRL-1573™) weremaintained in 50 ml Dulbecco's Modified Eagle Medium (Life Technologies)containing 100 units/mL of penicillin and 100 μg/mL of streptomycin aswell as fetal bovine serum at a dilution of 1:10. HEK cells were grownin incubators at 37° C./5% CO₂ and were transferred to a new 225 cm²culture flask (Thermo) every 3 to 4 days at passaging dilutions rangingfrom 1:5 to 1:8. 24 h prior to DNA transfections cells were plated on 2cm poly-D-lysine coated glass cover slips and maintained in 24 wellculture plates (Thermo) with 500 μl growth medium. 24 h prior torecordings, HEK cells were transfected with 1.6 μl plasmid DNA per wellusing 2 μl Lipofectamine 2000 (Life Technologies).

HEK Cell Electrophysiology

ChR-expressing cells were identified by eYFP fluorescence and recorded˜18 to 30 hours after transfection. The same equipment and methods asfor neurons was used. Measurements were conducted in voltage clamp atmembrane potentials between −75 and +55 mV. An external 3M CsCl agarbridge was used in all recordings. All constructs were characterizedusing the same internal and external recordings. All constructs werefirst characterized using the same internal and external solution as inneurons and were corrected for the corresponding junction potential, andstationary photocurrents were used for data analysis. The activationspectra for C1C2, iC1C2 and NpHR was determined by measuring stationaryphotocurrents at −75 mV in response to low light intensities at 0.65mW/mm² in order to prevent saturation. 20 nm bandbass filters (Thorlabs)were used to apply light at different wavelengths (in nm): 400, 420,440, 460, 470, 480, 490, 500, 520, 540, 560, 570, 580, 590, 600, 620,630, 650. All photocurrents were normalized to reference values at 470nm (C1C2 and iC1C2) or 570 nm (NpHR). Kinetics of channel closure werequantified by fitting photocurrents after light-off withmono-exponential functions in order to obtain corresponding tau_(off)values. Light sensitivity measurements were carried out at 470 nm (C1C2,iC1C2) or 560 nm (NpHR). Light was applied at intensities from 0.0021 to5 mW/mm² and normalized corresponding photocurrents to the value atmaximum light intensity.

Ion selectivities were determined by varying ion composition and pH ofthe internal and external solutions. External solutions contained (inmM) 2 CaCl₂, 2 MgCl₂, 120 NaCl, 120 CsCl or 120 Na-gluconate, 10 Citricacid/Na-citrate (pH 6) or 10 HEPES (pH 7.3) or 10 Tris (pH 9). Internalsolutions contained (in mM) 2 CaCl₂, 2 MgCl₂, 120 KCl, 120 CsCl or 120K-gluconate, 10 Citric acid/Na-citrate (pH 6) or 10 HEPES (pH 7.3) or 10Tris (pH 9). Junction potential was corrected for under each condition(in mV): KCl_(int)/NaCl_(ext)=4, KCl_(int)/CsCl_(ext=−0.6),KCl_(int)/NaGluc_(ext)=−6.2, KGluc_(int)/NaCl_(ext)=15.8,CsCl_(int)/NaCl_(ext)=4.6. The Nernst equation was used to determine theNernst potential for cations, Cl− and protons under each external andinternal ion composition and pH. An adapted Goldman-Hodgkin-Katzequation was used to calculate the ratio of proton to Cl⁻ permeabilityin iC1C2. Permeability for Na+ and K+ was assumed to be zero, whichresulted in:

$V_{rev} = {\frac{RT}{F}\ln \frac{{P_{H}\left\lbrack H^{+} \right\rbrack}_{ext} + {P_{Na}\left\lbrack {Na}^{+} \right\rbrack}_{ext} + {P_{K}\left\lbrack K^{+} \right\rbrack}_{ext} + {P_{Cl}\left\lbrack {Cl}^{-} \right\rbrack}_{int}}{{P_{H}\left\lbrack H^{+} \right\rbrack}_{int} + {P_{Na}\left\lbrack {Na}^{+} \right\rbrack}_{int} + {P_{K}\left\lbrack K^{+} \right\rbrack}_{int} + {P_{Cl}\left\lbrack {Cl}^{-} \right\rbrack}_{ext}}}$$V_{rev} = {\frac{RT}{F}\ln \; \frac{{P_{H}\left\lbrack H^{+} \right\rbrack}_{ext} + {P_{Cl}\left\lbrack {Cl}^{-} \right\rbrack}_{int}}{{P_{H}\left\lbrack H^{+} \right\rbrack}_{int} + {P_{Cl}\left\lbrack {Cl}^{-} \right\rbrack}_{ext}}}$$\alpha = \frac{P_{Cl}}{P_{H}}$$V_{rev} = {\frac{RT}{F}\ln \frac{\left\lbrack H^{+} \right\rbrack_{ext} + {\alpha \left\lbrack {Cl}^{-} \right\rbrack}_{int}}{\left\lbrack H^{+} \right\rbrack_{int} + {\alpha \left\lbrack {Cl}^{-} \right\rbrack}_{ext}}}$

R=Gas constant, F=Faraday constant, T=absolute temperature.

Statistical analysis was performed with a t-test or a two-way ANOVA, anda Mann-Whitney test for non-parametric data, using Origin8 (OriginLab)and Sigmaplot (SPSS) software. Data is presented as mean+s.e.m., anderror bars indicate s.e.m. p<0.05 is defined to be statisticallysignificant.

Example 1: Structure-Guided Screen for Light-Activated Anion ChannelProteins

A structure-guided screen was conducted by introducing singlesite-directed mutations into C1C2 (FIG. 1, Panel A). All variants wereexpressed in cultured rat hippocampal neurons and photocurrents weretested using a whole-cell patch-clamp to ensure proper function inneurons (external/internal [Cl⁻]: 147 mM/4 mM). Stationary photocurrentamplitudes were quantified across a range of holding potentials (FIG. 1,Panel B), with particular attention to V_(rev) to identify permeabilityvariants (FIG. 1, Panel C). C1C2 exhibited V_(rev) of −7 mV under theseconditions, typical for non-specific cation channels. Certain mutationswith powerful effects on V_(rev) displayed concomitant adverse effectson photocurrent sizes (e.g. E136R and E140K; FIG. 1, Panel B), and werenot studied further. More promising mutations, such as N297Q and H173R,exhibited both potent currents and altered V_(rev) (FIG. 1, Panel C),and were combined in a series of increasingly integrated mutations. The5-fold mutation T98S/E129S/E140S/E162S/T285N and 4-fold mutationV156K/H173R/V281K/N297Q both displayed prominently-shifted V_(rev) (inthe range of −40 mV) while maintaining functionality (FIG. 1, Panels Dand E).

These constructs were combined to generate a 9-fold mutated variant withcontiguous shifts in expected electrostatic potential distribution (FIG.2, Panel A, FIG. 5). The 9-fold variant was expressed in HEK-293 cellsto test both V_(rev) and permeability under controlled ion compositionand optimized voltage clamp settings (FIG. 2, Panel B). Photocurrentswere mapped over a broad range of membrane potentials (FIG. 2, Panel C)(from −75 mV to +55 mV; Methods). Under these conditions(external/internal [Cl⁻]: 147 mM/4 mM), the combined 9-fold mutationexhibited V_(rev) of −61 mV, far more negatively shifted than the C1C2backbone or either parental 4x or 5x construct (FIG. 2, Panel D).Despite this major change in functionality, both peak and stationaryphotocurrents remained fast and robust (predicting suitability foroptogenetics, especially since this channel could also recruit areduced-membrane resistance mechanism for spiking inhibition), and theoriginal blue light-activation spectrum of C1C2 was maintained, comparedwith the red-activation capability of the Cl⁻ pump eNpHR3.0 (thusmaintaining a separable channel for inhibitory control in optogeneticapplications; FIG. 2, Panel D). This 9-fold variant was termed “iC1C2”.

As the shifted V_(rev) could be attributable to increased K⁺ selectivityor a new Cl⁻ conductance, V_(rev) was measured under varying ioncompositions (corrected for the calculated junction potential arisingfrom each condition; Methods) in order to determine the specific ionselectivity of iC1C2. ChRs are highly permeable for protons andtypically show no selectivity between K⁺ and Na⁺. Therefore, with apipette solution composition of 120 mM KCl at pH 7.3 and a bath solutionof 120 mM NaCl also at pH 7.3, virtually no chemical gradient forpermeant ions would be expected, and indeed under these conditionsV_(rev) for both C1C2 and iC1C2 was −0 mV (FIG. 2, Panel F). Replacementof external KCl by CsCl would create a strong outward-directed gradientfor K⁺ ions, and, as expected under this condition, V_(rev) of C1C2dropped to −17.4 mV consistent with K⁺ as a major charge carrier.However, there was no such V_(rev) shift for iC1C2 (V_(rev)=−1 mV).These data do not support a hypothesis that iC1C2 achieves shiftedV_(rev) by increased K⁺ conductance, and, in fact, iC1C2 does notappreciably conduct K⁺ under these conditions (FIG. 2, Panel F). To testthe other possibility of new Cl⁻ conductivity, external Cl⁻ was replacedwith gluconate (for a chemical Cl⁻ gradient of 8 mM_(ext)/128 mM_(int)and shifting its Nernst potential to +71 mV). Despite this strongoutward-directed Cl⁻ gradient, C1C2 showed no shift in V_(rev) (0 mV),as expected since the native C1C2 does not conduct C1. In contrast,iC1C2 exhibited a positively shifted V_(rev) of +48 mV, revealing astrong contribution of Cl⁻ to the photocurrent (FIG. 2, Panel F).Finally, internal Cl⁻ was replaced with gluconate to create a stronginward-directed Cl⁻ gradient (128 mM [Cl⁻]_(ext)/8 mM [Cl⁻]_(int);V_(Nenst-Cl)=−71 mV). The resulting V_(rev) was −6 mV for C1C2 but −57mV for iC1C2, confirming a potent contribution from conducted Cl⁻ ionsto iC1C2 photocurrents (FIG. 2, Panel G).

Since the V_(rev) for iC1C2 was not identical to the calculatedV_(NernstCl), other ions, such as protons could be conducted as well. Toexplore this possibility in physiological Cl⁻ gradients, the protonconcentrations of internal and external solutions were altered whilemaintaining the inward-directed Cl⁻ chemical gradient (128 mM_(ex)/8mM_(int); V_(Nernst-Cl)=−71 mV) (FIG. 2, Panel H). The pH of externaland internal solutions was varied together (no proton chemical gradient;V_(Nernst-H+)=0 mV), and iC1C2 responses at physiological (7.3) and low(6.0) pH were measured, with matched internal/external protonconcentrations. The expectation was that at lower pH and more negativemembrane potential protons would contribute more to the iC1C2photocurrent, and thus positively shift V_(rev) towards the 0 mV Nernstpotential for protons. Surprisingly, the iC1C2 V_(rev) was morenegatively shifted at pH 6 compared to pH 7.3 (FIG. 2, Panel H),suggesting that iC1C2 conducts C1-even more robustly and maintains aprominently negative V_(rev) at lower pH values. Total iC1C2photocurrents were greater at lower pH values (FIG. 2, Panels I and J),consistent with a proton-enhanced Cl⁻ permeability. The ratio of Cl− toproton permeability was calculated at the different pH values (α=PCl/PH,see Methods). Indeed, at pH 6, the contribution of Cl⁻ to the overallcurrent was 35 times higher than at pH 7.3, suggesting that evenexcursions to lower pH as can happen during extreme neural activity willnot impair the important Cl⁻ conductance.

Example 2: Expression of Light-Activated Anion Channel Proteins inCultured Neurons

C1C2 and iC1C2 were expressed, each fused to enhanced yellow fluorescentprotein (eYFP), in cultured hippocampal neurons (FIG. 5). Mean restingpotentials were not different (C1C2: −65 mV; iC1C2: −69 mV) and inputresistances were in the expected range (above 200 MΩ) for bothconstructs. V_(rev) was determined (FIG. 3, Panel A), which for iC1C2(V_(rev)=−64 mV) was negatively shifted by 56 mV compared to C1C2(V_(rev)=−7 mV) (FIG. 3, Panel B). This V_(rev) of iC1C2 lies morenegative than the measured threshold for AP generation (VAP=−55 mV)(FIG. 3, Panel C). Consequently at V_(AP), in voltage clamp C1C2generated an inward-directed photocurrent of −475 pA, while iC1C2produced an outward-directed photocurrent of +42 pA; in current clamp,C1C2 depolarized neurons by +20 mV while iC1C2 hyperpolarized neurons by−3 mV (FIG. 3, Panel D). In addition, input resistance dropped by about50% during light in cells expressing iC1C2, indicating increased ionflux through membrane pores, and after light-off recovered to originallevels (FIG. 3, Panel E).

The iC1C2 input-resistance effects and iC1C2 membrane polarizationeffects that together would tend to maintain membrane potential belowspike-firing threshold (FIG. 3, Panel F), suggested utility inoptogenetic control of spiking. Indeed, optical activation of iC1C2sufficed to inhibit electrically-evoked spikes without exerting adepolarizing effect (FIG. 4, Panels A-C). To further explore theproperties of iC1C2, cysteine-167 was mutated to mimic step-functionmutations of channelrhodopsin, which decelerate channel closure andextend lifetime of the ion-conducting state; as a result, brief lightstimulation induces prolonged depolarization, and light sensitivity ofcells expressing these variants is greatly increased. The inhibitoryversions here define the SwiChR variants (for Step-waveform inhibitoryChR), including C167T (SwiChR_(CT)) and C167A (SwiChR_(CA)). SwiChR_(CT)was first expressed in HEK cells to determine channel kinetics andsensitivity. Both inward and outward-directed photocurrents werestabilized by orders-of-magnitude after light-off (FIG. 4, Panel D). Thetime-constant of channel closure (z off) for SwiChR_(CT) was 7.3 s,compared to 24 ms for the parent iC1C2 (FIG. 4, Panel E). Beyondstability, another feature of step-function variants is the ability toquickly convert to the closed state upon red-shifted light application,and indeed, SwiChR_(CT) channel closure was accelerated by applicationof 632 nm light (SwiChR_(CT) τ off-632=375 ms) (FIG. 4, Panel E).Another feature of step-function variants is increased light sensitivityof expressing cells, which effectively become photon integrators forlong light pulses. Indeed, SwiChR_(CT)-expressing cells showed a 25-foldincrease in light sensitivity compared to iC1C2, and a 200-fold increasecompared to the pump-based inhibitor NpHR (FIG. 4, Panel F). Similarresults were observed in neurons; SwiChR_(CT) generated outward currentat AP threshold in neurons with reversal potential of −61 mV and −67 mVfor SwiChR_(CA) (FIG. 4, Panel G; FIG. 7). This sufficed to stably andreversibly inhibit spiking (FIG. 4, Panel H; FIG. 7) with minimaldirectly-driven current (FIG. 4, Panel G; FIG. 7) or membrane potentialchange (FIG. 4, Panel H; FIG. 7), presenting desirable properties foroptogenetic investigation.

Structure-guided conversion of a cation-selective ChR into alight-activated Cl⁻ channel was demonstrated. The iC1C2 mechanismprovides more physiological inhibition that does not require a majormembrane potential change, and variants enable improvement of stabilityand light-sensitivity by orders of magnitude over existing inhibitorytools. Depolarization-block strategies with excitatory tools, whileuseful in some settings, may not reliably inhibit all targeted cells,since light intensities are highly variable in scattering tissue; incontrast, iC1C2-based tools can only depolarize membranes to V_(rev) of˜−64 mV (well below V_(AP)) and hyperpolarize when membrane potential isabove V_(rev) (FIG. 3, Panel F).

While aspects of final functionality arose by design (for example,removal of acidic residues and introduction of basic residues; FIG. 2,Panel A), other properties remain to be fully explored. The new Cl⁻permeability of iC1C2 not only provides an unexpectedly effectiveillustration of cation-channel to anion-channel conversion, but alsodemonstrates structure-guided design of ChRs for new classes offunctionality.

Example 3: Selective Interruption of Signaling Between Neurons

A network comprising two or more neurons that communicate with oneanother is identified. A target neuron in the network is identified anda nucleic acid encoding a subject light-activated anion channelpolypeptide is introduced into the target neuron. The introduction ofthe nucleic acid into the target neuron causes the light-activated anionchannel polypeptide to be expressed by the neuron and localized to theplasma membrane of the neuron. Subsequently, the target neuron, or aportion thereof, is illuminated with light of an activating wavelength.The illumination causes the light-activated anion channel polypeptide toopen its anion channel pore, which allows chloride anions to enter theneuron and hyperpolarize the plasma membrane. The hyperpolarization ofthe plasma membrane of the neuron inhibits signaling between two or moreneurons in the network by inhibiting the formation of an actionpotential in the target neuron.

Example 4: Treatment of Acute and Chronic Pain

One or more neurons that are associated with pain sensation by a subjectare identified. A nucleic acid encoding a subject light-activated anionchannel polypeptide is introduced into the one or more neuronsassociated with pain sensation. The introduction of the nucleic acidinto the one or more neurons causes the light-activated anion channelpolypeptide to be expressed by the neuron(s) and localized to the plasmamembrane of the neuron(s). Subsequently, the neuron(s), or a portionthereof, is illuminated with light of an activating wavelength. Theillumination causes the light-activated anion channel polypeptide toopen its anion channel pore, which allows chloride anions to enter theneuron and hyperpolarize the plasma membrane. The hyperpolarization ofthe plasma membrane of the neuron inhibits the formation of an actionpotential in the neuron, thereby blocking or alleviating the sensationof pain in the subject.

Example 5: Treatment of Cough by Inhibiting the Phrenic Nerve

A nucleic acid encoding a subject light-activated anion channelpolypeptide is introduced into the phrenic nerve of a subject having anurge to cough. The introduction of the nucleic acid into the phrenicnerve causes the light-activated anion channel polypeptide to beexpressed by the phrenic nerve and localized to the plasma membrane ofthe phrenic nerve. Subsequently, the phrenic nerve, or a portionthereof, is illuminated with light of an activating wavelength. Theillumination causes the light-activated anion channel polypeptide toopen its anion channel pore, which allows chloride anions to enter thephrenic nerve and hyperpolarize the plasma membrane. Thehyperpolarization of the plasma membrane of the phrenic nerve inhibitsthe formation of an action potential in the phrenic nerve, therebyblocking or reducing the subject's urge to cough.

Example 6: Expression of Light-Activated Anion Channel Proteins inCultured Neurons

ibC1C2_3.0 (SEQ ID NO: 96) and SwiChR_3.0 (SEQ ID NO: 108) constructswere tested in cultured neurons under physiological conditions with 4 mMand 12 mM internal chloride concentrations and compared to ibC1C2 (SEQID NO: 3) and SwiChR_CA (ibC1C2-C128A, SEQ ID NO: 15). At 4 mM internalchloride the reversal potential ofibC1C2_3.0 was −79 mV (±1 mV, standarderror of the mean (s.e.m.)) compared to −64 mV (±2 mV, s.e.m.) foribC1C2 (SEQ ID NO: 3) (FIG. 17, Panel A). This reflects an increasedselectivity for chloride ions in ibC1C2_3.0 (SEQ ID NO: 96).Furthermore, the photocurrents at the threshold for action potentialgeneration (VAP=−55 mV) are 408 pA (±60 pA, s.e.m.) for ibC1C2_3.0 (SEQID NO: 96) compared to 68 pA (±9 pA, s.e.m.) in ibC1C2 (SEQ ID NO: 3),which reflects an increased transport rate for chloride ions. At 12 mMinternal chloride concentration the reversal potentials were −66 mV (±1mV, s.e.m.) for ibC1C2_3.0 (SEQ ID NO: 96), −59 mV (±1 mV, s.e.m.) foribC1C2 (SEQ ID NO: 3), −65 mV (±1 mV, s.e.m.) for SwiChR_3.0 (SEQ ID NO:108) and −55 mV (±1 mV, s.e.m.) for SwiChR_CA (SEQ ID NO: 15) (FIG. 17,Panel B). The photocurrents at VAP were 171 pA (±31 pA, s.e.m.) foribC1C2_3.0 (SEQ ID NO: 96), 23 pA (±4 pA, s.e.m.) for ibC1C2 (SEQ ID NO:3), 91 pA (±14 pA, s.e.m.) for SwiChR_3.0 (SEQ ID NO: 108) and 21 pA forSwiChR_CA (SEQ ID NO: 15) (±7 pA, s.e.m.). Both measurements reflectthat the improved constructs have a higher chloride selectivity andconductivity at high internal chloride concentrations. As a result,ibC1C2_3.0 (SEQ ID NO: 96) and SwiChR_3.0 (SEQ ID NO: 108) have asignificantly higher probability to inhibit electrically evoked actionpotentials in neurons at low and high internal chloride concentrations.Cultured neurons were recorded in current clamp mode and actionpotentials were evoked by 30 ms long electrical pulses at 10 Hz for 6seconds. 1 s after the start of the stimulation, blue light (475 nm, 5mW/mm²) was applied for 4 seconds (FIG. 18, Panel A). At 4 mM chlorideconcentration, ibC1C2_3.0 (SEQ ID NO: 96) inhibited 100% of all actionpotentials whereas ibC1C2 (SEQ ID NO: 3) inhibited 58% (±12%, s.e.m.)(FIG. 18, Panel B). At 12 mM internal chloride the inhibitionprobability was 96% (±4%, s.e.m.) for ibC1C2_3.0 (SEQ ID NO: 96), 18%(±7%, s.e.m.) for ibC1C2 (SEQ ID NO: 3), 100% for SwiChR_3.0 (SEQ ID NO:108) and 45% for SwiChR_CA (SEQ ID NO: 15) (±13%, s.e.m.) (FIG. 18,Panel C). To demonstrate the capabilities of the improved constructs,even stronger inhibition protocols were applied. For example, ibC1C2_3.0(SEQ ID NO: 96)-expressing cultured neurons were stimulated for 12seconds at 20 Hz with 2 ms long electrical pulses to evoke actionpotentials (FIG. 19, Panel A). Blue light was applied for 10 s toinhibit spiking. At 12 mM chloride, the inhibition probability was 86%(±13%, s.e.m.) (FIG. 19, Panel C). Constructs which contain mutations atposition 128, such as SwiChR_CA (SEQ ID NO: 15) and SwiChR_3.0 (SEQ IDNO: 108) have a decelerated channel closure, i.e., the channel porestays open and conducts chloride ions for an extended period of timeafter blue light excitation has stopped. The channel can be immediatelyclosed by the application of red light (>600 nm). This allows forextended inhibition of neuronal activities upon brief blue lightapplications, which can be immediately recovered by red light pulses.This feature was demonstrated by inhibiting electrically evoked actionpotentials for up to 1 minute between the blue and red light pulses incultured neurons at 12 mM internal chloride (FIG. 18, Panel B, Panel C,FIG. 19, FIG. 20).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A light-activated polypeptide comprising an aminoacid sequence that is at least 58% identical to SEQ ID NOS: 1, 23, 34 or56, wherein the polypeptide functions as a light-activated anionchannel.
 2. A nucleic acid comprising a nucleotide sequence encoding thepolypeptide of claim 1, or a recombinant expression vector comprising anucleic acid comprising a nucleotide sequence encoding the polypeptideof claim
 1. 3. The polypeptide according to claim 1, wherein thepolypeptide functions as a light-activated chloride anion channel. 4.The polypeptide according to claim 1, wherein the polypeptide has anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 1 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q,relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
 5. Thepolypeptide according to claim 4, wherein the first 50 N-terminal aminoacid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82).
 6. The polypeptide according to claim 4,wherein the cysteine residue at position 167 is changed to a threonine,alanine or serine residue.
 7. The polypeptide according to claim 4,wherein the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue.
 8. The polypeptide accordingto claim 4, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 1-22.
 9. The polypeptide according toclaim 1, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acid substitutions selected from T98S, E129S, E140S, E162S, V156K,H173R, A285N, P281K and/or N297Q, relative to the amino acid sequence ofC1V1 (SEQ ID NO:80).
 10. The polypeptide according to claim 9, whereinthe first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 11. Thepolypeptide according to claim 9, wherein the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. 12.The polypeptide according to claim 9, wherein the aspartic acid residueat position 195 is changed to an alanine residue or an asparagineresidue.
 13. The polypeptide according to claim 9, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 34-55.
 14. The polypeptide according to claim 1, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T99S, E130S, E141S, E163S, V157K, H174R,A286N, P282K and/or N298Q, relative to the amino acid sequence of ReaChR(SEQ ID NO:81).
 15. The polypeptide according to claim 14, wherein thefirst 51 N-terminal amino acid residues are replaced by the followingamino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 16. The polypeptideaccording to claim 14, wherein the cysteine residue at position 168 ischanged to a threonine, alanine or serine residue.
 17. The polypeptideaccording to claim 14, wherein the aspartic acid residue at position 196is changed to an alanine residue or an asparagine residue.
 18. Thepolypeptide according to claim 14, wherein the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 56-77.
 19. Thepolypeptide according to claim 1, wherein the polypeptide has an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or is 100% identical tothe amino acid sequence provided in SEQ ID NO:23 and comprises 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions selected from A59S, E90S,E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q, relative to theamino acid sequence of ChR2 (SEQ ID NO:79).
 20. The polypeptideaccording to claim 19, wherein the cysteine residue at position 128 ischanged to a threonine, alanine or serine residue.
 21. The polypeptideaccording to claim 19, wherein the aspartic acid residue at position 156is changed to an alanine residue or an asparagine residue.
 22. Thepolypeptide according to claim 19, wherein the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 23-33.
 23. Apharmaceutical composition comprising the nucleic acid of claim 2 and apharmaceutically acceptable carrier.
 24. A cell comprising the nucleicacid of claim 2 or the recombinant expression vector of claim
 2. 25. Asystem for modulating the membrane potential of a cell, the systemcomprising: a nucleic acid encoding a polypeptide that comprises anamino acid sequence that is at least 58% identical to SEQ ID NOs: 1, 23,34 or 56, wherein the polypeptide functions as a light-activated anionchannel; and a device configured to illuminate a target location withlight.
 26. The system according to claim 25, wherein the polypeptidefunctions as a light-activated chloride ion channel.
 27. The systemaccording to claim 25, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO: 1 and comprises 1, 2, 3, 4,5, 6, 7, 8, or 9 amino acid substitutions selected from T98S, E129S,E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q, relative to theamino acid sequence of C1C2 (SEQ ID NO:78).
 28. The system according toclaim 27, wherein the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82).
 29. The system according to claim 27, wherein the cysteineresidue at position 167 is changed to a threonine, alanine or serineresidue.
 30. The system according to claim 27, wherein the aspartic acidresidue at position 195 is changed to an alanine residue or anasparagine residue.
 31. The system according to claim 27, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 1-22.
 32. The system according to claim 25, wherein the polypeptidehas an amino acid sequence that is at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO:34 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281K and/or N297Q,relative to the amino acid sequence of C1V1 (SEQ ID NO:80).
 33. Thesystem according to claim 32, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 34. The system according to claim 32, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 35. The system according to claim 32, wherein theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue.
 36. The system according to claim 32, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 34-55.
 37. The system according to claim 25, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T99S, E130S, E141S, E163S, V157K, H174R,A286N, P282K and/or N298Q, relative to the amino acid sequence of ReaChR(SEQ ID NO:81).
 38. The system according to claim 37, wherein the first51 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 39. The system according toclaim 37, wherein the cysteine residue at position 168 is changed to athreonine, alanine or serine residue.
 40. The system according to claim37, wherein the aspartic acid residue at position 196 is changed to analanine residue or an asparagine residue.
 41. The system according toclaim 37, wherein the polypeptide has an amino acid sequence as providedin any one of SEQ ID NOs: 56-77.
 42. The system according to claim 25,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R,V242K, T246N and/or N258Q, relative to the amino acid sequence of ChR2(SEQ ID NO:79).
 43. The system according to claim 42, wherein thecysteine residue at position 128 is changed to a threonine, alanine orserine residue.
 44. The system according to claim 42, wherein theaspartic acid residue at position 156 is changed to an alanine residueor an asparagine residue.
 45. The system according to claim 42, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 23-33.
 46. The system according to claim 25, wherein the deviceis configured to illuminate the target location with light having awavelength ranging from about 350 to about 750 nm.
 47. The systemaccording to claim 46, wherein the device is configured to illuminatethe target location with light having a wavelength ranging from about450 up to about 500 nm.
 48. The system according to claim 25, whereinthe device is configured to constantly illuminate the target locationwith light.
 49. The system according to claim 25, wherein the device isconfigured to illuminate the target location with pulses of light. 50.The system according to claim 25, wherein the device is configured tomodulate the wavelength and/or the intensity of the light.
 51. Thesystem according to claim 49, wherein the device is configured tomodulate the frequency and/or the duration of the pulses of light. 52.The system according to claim 25, wherein the device is configured toilluminate the target location in response to a user input.
 53. Thesystem according to 52, wherein the user input comprises: the wavelengthof light, the intensity of light, the duration of a light pulse, thefrequency of a light pulse, and/or the target location.
 54. The systemaccording to claim 25, wherein the device is adapted to be implanted ina subject.
 55. The system according to claim 25, wherein the targetlocation is: a cell, a portion of a cell, a plurality of cells, a bundleof nerve fibers, a neuromuscular junction, a central nervous system(CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomicalregion.
 56. A method for modulating the membrane potential of a cell inresponse to light, the method comprising: exposing a cell to light of anactivating wavelength, wherein the cell is genetically modified with anucleic acid encoding a polypeptide that comprises an amino acidsequence that is at least 58% identical to SEQ ID NOs: 1, 23, 34 or 56,wherein the polypeptide functions as a light-activated anion channel.57. The method according to claim 56, wherein the polypeptide has anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO:1 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q,relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
 58. Themethod according to claim 57, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 59. The method according to claim 57, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 60. The method according to claim 57, wherein theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue.
 61. The method according to claim 57, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 1-22.
 62. The method according to claim 56, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,A285N, P281K and/or N297Q, relative to the amino acid sequence of C1V1(SEQ ID NO:80).
 63. The method according to claim 62, wherein the first50 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 64. The method according toclaim 62, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 65. The method according to claim62, wherein the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue.
 66. The method according toclaim 62, wherein the polypeptide has an amino acid sequence as providedin any one of SEQ ID NOs: 34-55.
 67. The method according to claim 56,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T99S, E130S, E141S, E163S, V157K, H174R,A286N, P282K and/or N298Q, relative to the amino acid sequence of ReaChR(SEQ ID NO:81).
 68. The method according to claim 67, wherein the first51 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 69. The method according toclaim 67, wherein the cysteine residue at position 168 is changed to athreonine, alanine or serine residue.
 70. The method according to claim67, wherein the aspartic acid residue at position 196 is changed to analanine residue or an asparagine residue.
 71. The method according toclaim 67, wherein the polypeptide has an amino acid sequence as providedin any one of SEQ ID NOs: 56-77.
 72. The method according to claim 56,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R,V242K, T246N and/or N258Q, relative to the amino acid sequence of ChR2(SEQ ID NO:79).
 73. The method according to claim 72, wherein thecysteine residue at position 128 is changed to a threonine, alanine orserine residue.
 74. The method according to claim 72, wherein theaspartic acid residue at position 156 is changed to an alanine residueor an asparagine residue.
 75. The method according to claim 72, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 23-33.
 76. A method of treating a condition in a subject, themethod comprising: genetically modifying a target cell of the subjectwith a nucleic acid encoding a polypeptide that comprises an amino acidsequence that is at least 58% identical to SEQ ID NOs: 1, 23, 34 or 56,wherein the polypeptide functions as a light-activated anion channel;and exposing the target cell to light of an activating wavelength totreat the subject for the condition.
 77. The method according to claim76, wherein the polypeptide has an amino acid sequence that is at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 1 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acid substitutions selected from T98S, E129S, E140S, E162S, V156K,H173R, T285N, V281K and/or N297Q, relative to the amino acid sequence ofC1C2 (SEQ ID NO:78).
 78. The method according to claim 77, wherein thefirst 50 N-terminal amino acid residues are replaced by the followingamino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 79. The methodaccording to claim 77, wherein the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue.
 80. The methodaccording to claim 77, wherein the aspartic acid residue at position 195is changed to an alanine residue or an asparagine residue.
 81. Themethod according to claim 77, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 1-22.
 82. The methodaccording to claim 76, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO:34 and comprises 1, 2, 3, 4,5, 6, 7, 8, or 9 amino acid substitutions selected from T98S, E129S,E140S, E162S, V156K, H173R, A285N, P281K and/or N297Q, relative to theamino acid sequence of C1V1 (SEQ ID NO:80).
 83. The method according toclaim 82, wherein the first 50 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82).
 84. The method according to claim 82, wherein the cysteineresidue at position 167 is changed to a threonine, alanine or serineresidue.
 85. The method according to claim 82, wherein the aspartic acidresidue at position 195 is changed to an alanine residue or anasparagine residue.
 86. The method according to claim 82, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 34-55.
 87. The method according to claim 76, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T99S, E130S, E141S, E163S, V157K, H174R,A286N, P282K and/or N298Q, relative to the amino acid sequence of ReaChR(SEQ ID NO:81).
 88. The method according to claim 87, wherein the first51 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 89. The method according toclaim 87, wherein the cysteine residue at position 168 is changed to athreonine, alanine or serine residue.
 90. The method according to claim87, wherein the aspartic acid residue at position 196 is changed to analanine residue or an asparagine residue.
 91. The method according toclaim 87, wherein the polypeptide has an amino acid sequence as providedin any one of SEQ ID NOs: 56-77.
 92. The method according to claim 76,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R,V242K, T246N and/or N258Q, relative to the amino acid sequence of ChR2(SEQ ID NO:79).
 93. The method according to claim 92, wherein thecysteine residue at position 128 is changed to a threonine, alanine orserine residue.
 94. The method according to claim 92, wherein theaspartic acid residue at position 156 is changed to an alanine residueor an asparagine residue.
 95. The method according to claim 92, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 23-33.
 96. The method according to claim 76, wherein the targetcell is a nerve cell.
 97. A method of inhibiting the formation of anaction potential in a nerve cell or a portion thereof, the methodcomprising: genetically modifying the nerve cell with a nucleic acidencoding a polypeptide that comprises an amino acid sequence that is atleast 58% identical to SEQ ID NOs: 1, 23, 34 or 56, wherein thepolypeptide functions as a light-activated anion channel; and exposingat least a portion of the nerve cell to light of an activatingwavelength to inhibit the formation of an action potential in the nervecell or in a portion thereof.
 98. The method according to claim 97,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO: 1 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,T285N, V281K and/or N297Q, relative to the amino acid sequence of C1C2(SEQ ID NO:78).
 99. The method according to claim 98, wherein the first50 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 100. The method according toclaim 98, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 101. The method according to claim98, wherein the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue.
 102. The method according toclaim 98, wherein the polypeptide has an amino acid sequence as providedin any one of SEQ ID NOs: 1-22.
 103. The method according to claim 97,wherein the polypeptide has an amino acid sequence that is at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,A285N, P281K and/or N297Q, relative to the amino acid sequence of C1V1(SEQ ID NO:80).
 104. The method according to claim 103, wherein thefirst 50 N-terminal amino acid residues are replaced by the followingamino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 105. The methodaccording to claim 103, wherein the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue.
 106. The methodaccording to claim 103, wherein the aspartic acid residue at position195 is changed to an alanine residue or an asparagine residue.
 107. Themethod according to claim 103, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 34-55.
 108. The methodaccording to claim 97, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO:56 and comprises 1, 2, 3, 4,5, 6, 7, 8, or 9 amino acid substitutions selected from T99S, E130S,E141S, E163S, V157K, H174R, A286N, P282K and/or N298Q, relative to theamino acid sequence of ReaChR (SEQ ID NO:81).
 109. The method accordingto claim 108, wherein the first 51 N-terminal amino acid residues arereplaced by the following amino acids residues: MDYGGALSAVG (SEQ IDNO:82).
 110. The method according to claim 108, wherein the cysteineresidue at position 168 is changed to a threonine, alanine or serineresidue.
 111. The method according to claim 108, wherein the asparticacid residue at position 196 is changed to an alanine residue or anasparagine residue.
 112. The method according to claim 108, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 56-77.
 113. The method according to claim 97, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R,V242K, T246N and/or N258Q, relative to the amino acid sequence of ChR2(SEQ ID NO:79).
 114. The method according to claim 113, wherein thecysteine residue at position 128 is changed to a threonine, alanine orserine residue.
 115. The method according to claim 113, wherein theaspartic acid residue at position 156 is changed to an alanine residueor an asparagine residue.
 116. The method according to claim 113,wherein the polypeptide has an amino acid sequence as provided in any ofSEQ ID NOs: 23-33.
 117. A kit comprising: a nucleic acid encoding apolypeptide that is at least 58% identical to SEQ ID NOs: 1, 23, 34 or56, wherein the polypeptide functions as a light-activated anionchannel.
 118. The kit according to claim 117, wherein the polypeptidehas an amino acid sequence that is at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 1 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selectedfrom T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q,relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
 119. The kitaccording to claim 118, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 120. The kit according to claim 118, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 121. The kit according to claim 118, wherein theaspartic acid residue at position 195 is changed to an alanine residueor an asparagine residue.
 122. The kit according to claim 118, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 1-22.
 123. The kit according to claim 117, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:34 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions selected from T98S, E129S, E140S, E162S, V156K, H173R,A285N, P281K and/or N297Q, relative to the amino acid sequence of C1V1(SEQ ID NO:80).
 124. The kit according to claim 123, wherein the first50 N-terminal amino acid residues are replaced by the following aminoacids residues: MDYGGALSAVG (SEQ ID NO:82).
 125. The kit according toclaim 123, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 126. The kit according to claim123, wherein the aspartic acid residue at position 195 is changed to analanine residue or an asparagine residue.
 127. The kit according toclaim 123, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 34-55.
 128. The kit according toclaim 117, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO:56 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acid substitutions selected from T99S, E130S, E141S, E163S, V157K,H174R, A286N, P282K and/or N298Q, relative to the amino acid sequence ofReaChR (SEQ ID NO:81).
 129. The kit according to claim 128, wherein thefirst 51 N-terminal amino acid residues are replaced by the followingamino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 130. The kit accordingto claim 128, wherein the cysteine residue at position 168 is changed toa threonine, alanine or serine residue.
 131. The kit according to claim128, wherein the aspartic acid residue at position 196 is changed to analanine residue or an asparagine residue.
 132. The kit according toclaim 128, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 56-77.
 133. The kit according toclaim 117, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO:23 and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9amino acid substitutions selected from A59S, E90S, E101S, E123S, Q117K,H134R, V242K, T246N and/or N258Q, relative to the amino acid sequence ofChR2 (SEQ ID NO:79).
 134. The kit according to claim 133, wherein thecysteine residue at position 128 is changed to a threonine, alanine orserine residue.
 135. The kit according to claim 133, wherein theaspartic acid residue at position 156 is changed to an alanine residueor an asparagine residue.
 136. The kit according to claim 133, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 23-33.
 137. The kit according to claim 117, further comprising adevice configured to illuminate a target location with a light.
 138. Thekit according to claim 137, wherein the device is configured toilluminate the target location with light having a wavelength rangingfrom about 350 to about 750 nm.
 139. The kit according to claim 138,wherein the device is configured to illuminate the target location withlight having a wavelength ranging from about 450 up to about 500 nm.140. The kit according to claim 137, wherein the device is configured toconstantly illuminate the target location with a light.
 141. The kitaccording to claim 137, wherein the device is configured to illuminatethe target location with pulses of light.
 142. The kit according toclaim 137, wherein the device is configured to modulate the wavelengthand/or the intensity of the light.
 143. The kit according to claim 141,wherein the device is configured to modulate the frequency and/orduration of the pulses of light.
 144. The kit according to claim 137,wherein the device is configured to illuminate the target location inresponse to a user input.
 145. The kit according to claim 144, whereinthe user input comprises: the wavelength of light, the intensity oflight, the duration of a light pulse, the frequency of a light pulse,and/or the target location to be illuminated by the light.
 146. The kitaccording to claim 137, wherein the device is adapted to be implanted ina subject.
 147. The kit according to claim 137, wherein the targetlocation is: a cell, a portion of a cell, a plurality of cells, a bundleof nerve fibers, a neuromuscular junction, a central nervous system(CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomicalregion.
 148. A light-activated polypeptide comprising an amino acidsequence that is at least 60% identical to SEQ ID NOS: 94, 116, 127 or149, wherein the polypeptide functions as a light-activated anionchannel.
 149. A nucleic acid comprising a nucleotide sequence encodingthe polypeptide of claim 148, or a recombinant expression vectorcomprising a nucleic acid comprising a nucleotide sequence encoding thepolypeptide of claim
 148. 150. The polypeptide according to claim 148,wherein the polypeptide functions as a light-activated chloride anionchannel.
 151. The polypeptide according to claim 148, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:94 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T98S, E122N, E129Q, E140S, V156R, E162S,V281R, T285N, N297Q and/or E312S, relative to the amino acid sequence ofC1C2 (SEQ ID NO:78).
 152. The polypeptide according to claim 151,wherein the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 153. Thepolypeptide according to claim 151, wherein the cysteine residue atposition 167 is changed to a threonine, alanine or serine residue. 154.The polypeptide according to claim 151, wherein the aspartic acidresidue at position 195 is changed to an alanine residue, an asparagineresidue, or a cysteine residue.
 155. The polypeptide according to claim152, wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 156. Thepolypeptide according to claim 151, wherein the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 94-115.
 157. Thepolypeptide according to claim 148, wherein the polypeptide has an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or is 100% identical tothe amino acid sequence provided in SEQ ID NO: 127 and comprises 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions selected from T98S,E122N, E129Q, E140S, V156R, E162S, P281R, A285N, N297Q and/or E312S,relative to the amino acid sequence of C1V1 (SEQ ID NO:80).
 158. Thepolypeptide according to claim 157, wherein the first 50 N-terminalamino acid residues are replaced by the following amino acids residues:MDYGGALSAVG (SEQ ID NO:82).
 159. The polypeptide according to claim 157,wherein the cysteine residue at position 167 is changed to a threonine,alanine or serine residue.
 160. The polypeptide according to claim 157,wherein the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 161. Thepolypeptide according to claim 158, wherein the aspartic acid residue atposition 156 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 162. The polypeptide according to claim 157, whereinthe polypeptide has an amino acid sequence as provided in any one of SEQID NOs: 127-148.
 163. The polypeptide according to claim 148, whereinthe polypeptide has an amino acid sequence that is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or is 100% identical to the amino acid sequence provided inSEQ ID NO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T99S, E123N, E130Q, E141S, V157R, E163S,P282R, A286N, N298Q and/or E313S, relative to the amino acid sequence ofReaChR (SEQ ID NO:81).
 164. The polypeptide according to claim 163,wherein the first 51 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 165. Thepolypeptide according to claim 163, wherein the cysteine residue atposition 168 is changed to a threonine, alanine or serine residue. 166.The polypeptide according to claim 163, wherein the aspartic acidresidue at position 196 is changed to an alanine residue, an asparagineresidue, or a cysteine residue.
 167. The polypeptide according to claim164, wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 168. Thepolypeptide according to claim 163, wherein the polypeptide has an aminoacid sequence as provided in any one of SEQ ID NOs: 149-170.
 169. Thepolypeptide according to claim 148, wherein the polypeptide has an aminoacid sequence that is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or is 100% identical tothe amino acid sequence provided in SEQ ID NO: 116 and comprises 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions selected from A59S,E83N, E90Q, E101S, Q117R, E123S, V242R, T246N, N258Q and/or E273S,relative to the amino acid sequence of ChR2 (SEQ ID NO:79).
 170. Thepolypeptide according to claim 169, wherein the cysteine residue atposition 128 is changed to a threonine, alanine or serine residue. 171.The polypeptide according to claim 169, wherein the aspartic acidresidue at position 156 is changed to an alanine residue, an asparagineresidue, or a cysteine residue.
 172. The polypeptide according to claim169, wherein the polypeptide has an amino acid sequence as provided inany one of SEQ ID NOs: 116-126.
 173. A pharmaceutical compositioncomprising the nucleic acid of claim 149 and a pharmaceuticallyacceptable carrier.
 174. A cell comprising the nucleic acid of claim 149or the recombinant expression vector of claim
 149. 175. A system formodulating the membrane potential of a cell, the system comprising: anucleic acid encoding a polypeptide that comprises an amino acidsequence that is at least 60% identical to SEQ ID NOs: 94, 116, 127 or149, wherein the polypeptide functions as a light-activated anionchannel; and a device configured to illuminate a target location withlight.
 176. The system according to claim 175, wherein the polypeptidefunctions as a light-activated chloride ion channel.
 177. The systemaccording to claim 175, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO:94 and comprises 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid substitutions selected from T98S, E122N,E129Q, E140S, V156R, E162S, V281R, T285N, N297Q and/or E312S, relativeto the amino acid sequence of C1C2 (SEQ ID NO:78).
 178. The systemaccording to claim 177, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 179. The system according to claim 177, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 180. The system according to claim 177, wherein theaspartic acid residue at position 195 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 181. The system accordingto claim 178, wherein the aspartic acid residue at position 156 ischanged to an alanine residue, an asparagine residue, or a cysteineresidue.
 182. The system according to claim 177, wherein the polypeptidehas an amino acid sequence as provided in any one of SEQ ID NOs: 94-115.183. The system according to claim 175, wherein the polypeptide has anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80).
 184. The system according to claim 183, wherein the first 50N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82).
 185. The system according to claim183, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 186. The system according to claim183, wherein the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 187. Thesystem according to claim 184, wherein the aspartic acid residue atposition 156 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 188. The system according to claim 183, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148.
 189. The system according to claim 175, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T99S, E123N, E130Q, E141S, V157R, E163S,P282R, A286N, N298Q and/or E313S, relative to the amino acid sequence ofReaChR (SEQ ID NO:81).
 190. The system according to claim 189, whereinthe first 51 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 191. Thesystem according to claim 189, wherein the cysteine residue at position168 is changed to a threonine, alanine or serine residue.
 192. Thesystem according to claim 189, wherein the aspartic acid residue atposition 196 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 193. The system according to claim 190, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 194. The system accordingto claim 189, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 149-170.
 195. The system according toclaim 175, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 116 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from A59S, E83N, E90Q, E101S, Q117R,E123S, V242R, T246N, N258Q and/or E273S, relative to the amino acidsequence of ChR2 (SEQ ID NO:79).
 196. The system according to claim 195,wherein the cysteine residue at position 128 is changed to a threonine,alanine or serine residue.
 197. The system according to claim 195,wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 198. Thesystem according to claim 195, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 116-126.
 199. The systemaccording to claim 175, wherein the device is configured to illuminatethe target location with light having a wavelength ranging from about350 to about 750 nm.
 200. The system according to claim 199, wherein thedevice is configured to illuminate the target location with light havinga wavelength ranging from about 450 up to about 500 nm.
 201. The systemaccording to claim 175, wherein the device is configured to constantlyilluminate the target location with light.
 202. The system according toclaim 175, wherein the device is configured to illuminate the targetlocation with pulses of light.
 203. The system according to claim 175,wherein the device is configured to modulate the wavelength and/or theintensity of the light.
 204. The system according to claim 202, whereinthe device is configured to modulate the frequency and/or the durationof the pulses of light.
 205. The system according to claim 175, whereinthe device is configured to illuminate the target location in responseto a user input.
 206. The system according to claim 205, wherein theuser input comprises: the wavelength of light, the intensity of light,the duration of a light pulse, the frequency of a light pulse, and/orthe target location.
 207. The system according to claim 175, wherein thedevice is adapted to be implanted in a subject.
 208. The systemaccording to claim 175, wherein the target location is: a cell, aportion of a cell, a plurality of cells, a bundle of nerve fibers, aneuromuscular junction, a central nervous system (CNS) tissue, aperipheral nervous system (PNS) tissue, or an anatomical region.
 209. Amethod for modulating the membrane potential of a cell in response tolight, the method comprising: exposing a cell to light of an activatingwavelength, wherein the cell is genetically modified with a nucleic acidencoding a polypeptide that comprises an amino acid sequence that is atleast 58% identical to SEQ ID NOs: 94, 116, 127 or 149, wherein thepolypeptide functions as a light-activated anion channel.
 210. Themethod according to claim 209, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO:94 and comprises 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid substitutions selected from T98S, E122N,E129Q, E140S, V156R, E162S, V281R, T285N, N297Q and/or E312S, relativeto the amino acid sequence of C1C2 (SEQ ID NO:78).
 211. The methodaccording to claim 210, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 212. The method according to claim 210, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 213. The method according to claim 210, wherein theaspartic acid residue at position 195 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 214. The method accordingto claim 211, wherein the aspartic acid residue at position 156 ischanged to an alanine residue, an asparagine residue, or a cysteineresidue.
 215. The method according to claim 210, wherein the polypeptidehas an amino acid sequence as provided in any one of SEQ ID NOs: 94-115.216. The method according to claim 209, wherein the polypeptide has anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80).
 217. The method according to claim 216, wherein the first 50N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82).
 218. The method according to claim216, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 219. The method according to claim216, wherein the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 220. Themethod according to claim 217, wherein the aspartic acid residue atposition 156 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 221. The method according to claim 216, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148.
 222. The method according to claim 210, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T99S, E123N, E130Q, E141S, V157R, E163S,P282R, A286N, N298Q and/or E313S, relative to the amino acid sequence ofReaChR (SEQ ID NO:81).
 223. The method according to claim 222, whereinthe first 51 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 224. Themethod according to claim 222, wherein the cysteine residue at position168 is changed to a threonine, alanine or serine residue.
 225. Themethod according to claim 222, wherein the aspartic acid residue atposition 196 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 226. The method according to claim 223, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 227. The method accordingto claim 222, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 149-170.
 228. The method according toclaim 209, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 116 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from A59S, E83N, E90Q, E101S, Q117R,E123S, V242R, T246N, N258Q and/or E273S, relative to the amino acidsequence of ChR2 (SEQ ID NO:79).
 229. The method according to claim 228,wherein the cysteine residue at position 128 is changed to a threonine,alanine or serine residue.
 230. The method according to claim 228,wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 231. Themethod according to claim 228, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 116-126.
 232. A method oftreating a condition in a subject, the method comprising: geneticallymodifying a target cell of the subject with a nucleic acid encoding apolypeptide that comprises an amino acid sequence that is at least 60%identical to SEQ ID NOs: 94, 116, 127 or 149, wherein the polypeptidefunctions as a light-activated anion channel; and exposing the targetcell to light of an activating wavelength to treat the subject for thecondition.
 233. The method according to claim 232, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO:94 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T98S, E122N, E129Q, E140S, V156R, E162S,V281R, T285N, N297Q and/or E312S, relative to the amino acid sequence ofC1C2 (SEQ ID NO:78).
 234. The method according to claim 233, wherein thefirst 50 N-terminal amino acid residues are replaced by the followingamino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 235. The methodaccording to claim 233, wherein the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue.
 236. The methodaccording to claim 233, wherein the aspartic acid residue at position195 is changed to an alanine residue, an asparagine residue, or acysteine residue.
 237. The method according to claim 234, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 238. The method accordingto claim 233, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 94-115.
 239. The method according toclaim 232, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 127 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T98S, E122N, E129Q, E140S, V156R,E162S, P281R, A285N, N297Q and/or E312S, relative to the amino acidsequence of C1V1 (SEQ ID NO:80).
 240. The method according to claim 239,wherein the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 241. Themethod according to claim 239, wherein the cysteine residue at position167 is changed to a threonine, alanine or serine residue.
 242. Themethod according to claim 239, wherein the aspartic acid residue atposition 195 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 243. The method according to claim 240, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 244. The method accordingto claim 239, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 127-148.
 245. The method according toclaim 232, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T99S, E123N, E130Q, E141S, V157R,E163S, P282R, A286N, N298Q and/or E313S, relative to the amino acidsequence of ReaChR (SEQ ID NO:81).
 246. The method according to claim245, wherein the first 51 N-terminal amino acid residues are replaced bythe following amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 247. Themethod according to claim 245, wherein the cysteine residue at position168 is changed to a threonine, alanine or serine residue.
 248. Themethod according to claim 245, wherein the aspartic acid residue atposition 196 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 249. The method according to claim 246, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 250. The method accordingto claim 245, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 149-170.
 251. The method according toclaim 232, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 116 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from A59S, E83N, E90Q, E101S, Q117R,E123S, V242R, T246N, N258Q and/or E273S, relative to the amino acidsequence of ChR2 (SEQ ID NO:79).
 252. The method according to claim 251,wherein the cysteine residue at position 128 is changed to a threonine,alanine or serine residue.
 253. The method according to claim 251,wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 254. Themethod according to claim 251, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 116-126.
 255. The methodaccording to claim 232, wherein the target cell is a nerve cell.
 256. Amethod of inhibiting the formation of an action potential in a nervecell or a portion thereof, the method comprising: genetically modifyingthe nerve cell with a nucleic acid encoding a polypeptide that comprisesan amino acid sequence that is at least 60% identical to SEQ ID NOs: 94,116, 127 or 149, wherein the polypeptide functions as a light-activatedanion channel; and exposing at least a portion of the nerve cell tolight of an activating wavelength to inhibit the formation of an actionpotential in the nerve cell or in a portion thereof.
 257. The methodaccording to claim 256, wherein the polypeptide has an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or is 100% identical to theamino acid sequence provided in SEQ ID NO:94 and comprises 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid substitutions selected from T98S, E122N,E129Q, E140S, V156R, E162S, V281R, T285N, N297Q and/or E312S, relativeto the amino acid sequence of C1C2 (SEQ ID NO:78).
 258. The methodaccording to claim 257, wherein the first 50 N-terminal amino acidresidues are replaced by the following amino acids residues: MDYGGALSAVG(SEQ ID NO:82).
 259. The method according to claim 257, wherein thecysteine residue at position 167 is changed to a threonine, alanine orserine residue.
 260. The method according to claim 257, wherein theaspartic acid residue at position 195 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 261. The method accordingto claim 258, wherein the aspartic acid residue at position 156 ischanged to an alanine residue, an asparagine residue, or a cysteineresidue.
 262. The method according to claim 257, wherein the polypeptidehas an amino acid sequence as provided in any one of SEQ ID NOs: 94-115.263. The method according to claim 256, wherein the polypeptide has anamino acid sequence that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or is 100%identical to the amino acid sequence provided in SEQ ID NO: 127 andcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsselected from T98S, E122N, E129Q, E140S, V156R, E162S, P281R, A285N,N297Q and/or E312S, relative to the amino acid sequence of C1V1 (SEQ IDNO:80).
 264. The method according to claim 263, wherein the first 50N-terminal amino acid residues are replaced by the following amino acidsresidues: MDYGGALSAVG (SEQ ID NO:82).
 265. The method according to claim263, wherein the cysteine residue at position 167 is changed to athreonine, alanine or serine residue.
 266. The method according to claim263, wherein the aspartic acid residue at position 195 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 267. Themethod according to claim 264, wherein the aspartic acid residue atposition 156 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 268. The method according to claim 263, wherein thepolypeptide has an amino acid sequence as provided in any one of SEQ IDNOs: 127-148.
 269. The method according to claim 256, wherein thepolypeptide has an amino acid sequence that is at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or is 100% identical to the amino acid sequence provided in SEQ IDNO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions selected from T99S, E123N, E130Q, E141S, V157R, E163S,P282R, A286N, N298Q and/or E313S, relative to the amino acid sequence ofReaChR (SEQ ID NO:81).
 270. The method according to claim 269, whereinthe first 51 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 271. Themethod according to claim 269, wherein the cysteine residue at position168 is changed to a threonine, alanine or serine residue.
 272. Themethod according to claim 269, wherein the aspartic acid residue atposition 196 is changed to an alanine residue, an asparagine residue, ora cysteine residue.
 273. The method according to claim 270, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 274. The method accordingto claim 269, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 149-170.
 275. The method according toclaim 256, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 116 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from A59S, E83N, E90Q, E101S, Q117R,E123S, V242R, T246N, N258Q and/or E273S, relative to the amino acidsequence of ChR2 (SEQ ID NO:79).
 276. The method according to claim 275,wherein the cysteine residue at position 128 is changed to a threonine,alanine or serine residue.
 277. The method according to claim 275,wherein the aspartic acid residue at position 156 is changed to analanine residue, an asparagine residue, or a cysteine residue.
 278. Themethod according to claim 275, wherein the polypeptide has an amino acidsequence as provided in any of SEQ ID NOs: 116-126.
 279. A kitcomprising: a nucleic acid encoding a polypeptide that is at least 60%identical to SEQ ID NOs: 94, 116, 127 or 149, wherein the polypeptidefunctions as a light-activated anion channel.
 280. The kit according toclaim 279, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO:94 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T98S, E122N, E129Q, E140S, V156R,E162S, V281R, T285N, N297Q and/or E312S, relative to the amino acidsequence of C1C2 (SEQ ID NO:78).
 281. The kit according to claim 280,wherein the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 282. The kitaccording to claim 280, wherein the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue.
 283. The kitaccording to claim 280, wherein the aspartic acid residue at position195 is changed to an alanine residue, an asparagine residue, or acysteine residue.
 284. The kit according to claim 281, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 285. The kit according toclaim 280, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 94-115.
 286. The kit according toclaim 279, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 127 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T98S, E122N, E129Q, E140S, V156R,E162S, P281R, A285N, N297Q and/or E312S, relative to the amino acidsequence of C1V1 (SEQ ID NO:80).
 287. The kit according to claim 286,wherein the first 50 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 288. The kitaccording to claim 286, wherein the cysteine residue at position 167 ischanged to a threonine, alanine or serine residue.
 289. The kitaccording to claim 286, wherein the aspartic acid residue at position195 is changed to an alanine residue, an asparagine residue, or acysteine residue.
 290. The kit according to claim 287, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 291. The kit according toclaim 286, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 127-148.
 292. The kit according toclaim 279, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 149 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from T99S, E123N, E130Q, E141S, V157R,E163S, P282R, A286N, N298Q and/or E313S, relative to the amino acidsequence of ReaChR (SEQ ID NO:81).
 293. The kit according to claim 292,wherein the first 51 N-terminal amino acid residues are replaced by thefollowing amino acids residues: MDYGGALSAVG (SEQ ID NO:82).
 294. The kitaccording to claim 292, wherein the cysteine residue at position 168 ischanged to a threonine, alanine or serine residue.
 295. The kitaccording to claim 292, wherein the aspartic acid residue at position196 is changed to an alanine residue, an asparagine residue, or acysteine residue.
 296. The kit according to claim 293, wherein theaspartic acid residue at position 156 is changed to an alanine residue,an asparagine residue, or a cysteine residue.
 297. The kit according toclaim 292, wherein the polypeptide has an amino acid sequence asprovided in any one of SEQ ID NOs: 149-170.
 298. The kit according toclaim 279, wherein the polypeptide has an amino acid sequence that is atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceprovided in SEQ ID NO: 116 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions selected from A59S, E83N, E90Q, E101S, Q117R,E123S, V242R, T246N, N258Q and/or E273S, relative to the amino acidsequence of ChR2 (SEQ ID NO:79).
 299. The kit according to claim 298,wherein the cysteine residue at position 128 is changed to a threonine,alanine or serine residue.
 300. The kit according to claim 298, whereinthe aspartic acid residue at position 156 is changed to an alanineresidue, an asparagine residue, or a cysteine residue.
 301. The kitaccording to claim 298, wherein the polypeptide has an amino acidsequence as provided in any one of SEQ ID NOs: 116-126.
 302. The kitaccording to claim 279, further comprising a device configured toilluminate a target location with a light.
 303. The kit according toclaim 302, wherein the device is configured to illuminate the targetlocation with light having a wavelength ranging from about 350 to about750 nm.
 304. The kit according to claim 303, wherein the device isconfigured to illuminate the target location with light having awavelength ranging from about 450 up to about 500 nm.
 305. The kitaccording to claim 302, wherein the device is configured to constantlyilluminate the target location with a light.
 306. The kit according toclaim 302, wherein the device is configured to illuminate the targetlocation with pulses of light.
 307. The kit according to claim 302,wherein the device is configured to modulate the wavelength and/or theintensity of the light.
 308. The kit according to claim 302, wherein thedevice is configured to modulate the frequency and/or duration of thepulses of light.
 309. The kit according to claim 302, wherein the deviceis configured to illuminate the target location in response to a userinput.
 310. The kit according to claim 309, wherein the user inputcomprises: the wavelength of light, the intensity of light, the durationof a light pulse, the frequency of a light pulse, and/or the targetlocation to be illuminated by the light.
 311. The kit according to claim302, wherein the device is adapted to be implanted in a subject. 312.The kit according to claim 302, wherein the target location is: a cell,a portion of a cell, a plurality of cells, a bundle of nerve fibers, aneuromuscular junction, a central nervous system (CNS) tissue, aperipheral nervous system (PNS) tissue, or an anatomical region.